Jens Høiriis Nielsen

Truncated glucagon-like peptide-1 (proglucagon 78–107 amide), an intestinal insulin-releasing peptide, has specific receptors on rat insulinoma cells (RIN 5AH)

We studied binding of 125I‐labelled truncated‐glucagon‐like peptide‐1 (proglucagon 78–107 amide) to a cloned rat insulin‐producing cell line, RIN 5AH, in monolayer culture. Interaction of the peptide with pancreatic insulinoma cells was saturable and time dependent. Half‐maximal binding was obtained when the cells were incubated in the presence of 3.3 × 10−9 mol/l unlabelled truncated‐glucagon‐like peptide‐1 (proglucagon 78–107 amide). Neither glucagon, full‐length glucagon‐like peptide‐1 (proglucagon 72–107 amide) nor gastric inhibitory peptide competed for binding in concentrations up to 10−6 mol/l.

Affinity-purified human Interleukin I is cytotoxic to isolated islets of Langerhans

SummaryAddition of highly purified human Interleukin-1 to the culture medium of isolated rat islets of Langerhans for 6 days led to 88% inhibition of glucose-induced insulin-release, reduction of islet contents of insulin and glucagon to 31% and 8% respectively, and disintegration of the islets. These effects were dose-dependent and reproducible when using three different Interleukin-1 preparations. Highly purified human Interleukin-2, Lymphotoxin, Leucocyte Migration Inhibitory Factor and Macrophage Migration Inhibitory Factor were ineffective. These findings suggest that Interleukin-1 may play an important role in the molecular mechanisms underlying autoimmune B-cell destruction leading to Type 1 (insulin-dependent) diabetes mellitus.

Glucagon-like peptide 1 (7-36) amide stimulates exocytosis in human pancreatic beta-cells by both proximal and distal regulatory steps in stimulus-secretion coupling.

The effect of glucagon-like peptide 1(7–36) amide [GLP-l(7–36) amide] on membrane potential, wholecell ATP-sensitive potassium channel (KATP) and Ca2+ currents, cytoplasmic Ca2+ concentration, and exocytosis was explored in single human β-cells. GLP-1(7–36) amide induced membrane depolarization that was associated with inhibition of whole-cell KATP current. In addition, GLP-l(7–36) amide (and forskolin) produced greater than fourfold potentiation of Ca2+-dependent exocytosis. The latter effect resulted in part (40%) from acceleration of Ca2+ influx through voltage-dependent (L-type) Ca2+ channels. More importantly, GLP- 1(7–36) amide (via generation of cyclic AMP and activation of protein kinase A) potentiated exocytosis at a site distal to a rise in the cytoplasmic Ca2+ concentration. Photorelease of caged cAMP produced a two- to threefold potentiation of exocytosis when the cytoplasmic Ca2+ concentrations were clamped at >170 nmol/1. The effect of GLP-1(7–36) amide was antagonized by the islet hormone somatostatin. Similar effects on membrane potential, ion conductances, and exocytosis were observed with glucose-dependent insulinotropic polypeptide (GIP), the second major incretin. The present data suggest that the strong insulinotropic action of GLP-1(7–36) amide and GIP in humans results from its interaction with several proximal as well as distal important regulatory steps in the stimulus-secretion coupling.

Suppressor of cytokine signaling 3 (SOCS-3) protects β-cells against interleukin-1β- and interferon-γ-mediated toxicity

Suppressor of cytokine signaling 3 (SOCS-3) is a negative feedback regulator of IFN-γ signaling, shown up-regulated in mouse bone marrow cells by the proinflammatory cytokines interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and IFN-γ. IL-1β and IFN-γ alone, or potentiated by TNF-α, are cytotoxic to the insulin producing pancreatic β-cells and β-cell lines in vitro and suggested to contribute to the specific β-cell destruction in Type-1 diabetes mellitus (T1DM). Using a doxycycline-inducible SOCS-3 expression system in the rat β-cell line INS-1, we demonstrate that the toxic effect of both IL-1β or IFN-γ at concentrations that reduced the viability by 50% over 3 days, was fully preventable when SOCS-3 expression was turned on in the cells. At cytokine concentrations or combinations more toxic to the cells, SOCS-3 overexpression yielded a partial protection. Whereas SOCS-3-mediated inhibition of IFN-γ signaling is described in other cell systems, SOCS-3 mediated inhibition of IL-1β signaling has not previously been described. In addition we show that SOCS-3 prevention of IL-1β-induced toxicity is accompanied by inhibited transcription of the inducible nitric oxide synthase (iNOS) by 80%, resulting in 60% decreased formation of the toxic nitric oxide (NO). Analysis of isolated native rat islets exposed to IL-1β revealed a naturally occurring but delayed up-regulated SOCS-3 transcription. Influencing SOCS-3 expression thus represents an approach for affecting cytokine-induced signal transduction at a proximal step in the signal cascade, potentially useful in future therapies aimed at reducing the destructive potential of β-cell cytotoxic cytokines in T1DM, as well as other cytokine-dependent diseases.

Beta cell proliferation and growth factors

Formation of new beta cells can take place by two pathways: replication of already differentiated beta cells or neogenesis from putative islet stem cells. Under physiological conditions both processes are most pronounced during the fetal and neonatal development of the pancreas. In adulthood little increase in the beta cell number seems to occur. In pregnancy, however, a marked hyperplasia of the beta cells is observed both in rodents and man. Increased mitotic activity has been seen both in vivo and in vitro in islets exposed to placental lactogen (PL), prolactin (PRL) and growth hormone (GH). Receptors for both GH and PRL are expressed in islet cells and are upregulated during pregnancy. By mutational analysis we have identified different functional domains of the cytoplasmic part of the GH receptor. Thus the mitotic signaling only requires the membrane proximal part of the receptor and activation of the tyrosine kinase JAK2 and the transcription factors STAT1 and 3. The activation of the insulin gene however also requires the distal part of the receptor and activation of calcium uptake and STAT5. In order to identify putative autocrine growth factors or targets for growth factors we have cloned a novel GH/PRL stimulated rat islet gene product, Pref-1 (preadipocyte factor-1). This protein contains six EGF-like motifs and may play a role both in embryonic pancreas differentiation and in beta cell growth and function. In summary, the increasing knowledge about the mechanisms involved in beta cell differentiation and proliferation may lead to new ways of forming beta cells for treatment of diabetes in man.

Identification of Tyrosine Residues in the Intracellular Domain of the Growth Hormone Receptor Required for Transcriptional Signaling and Stat5 Activation

The binding of growth hormone (GH) to its receptor results in its dimerization followed by activation of Jak2 kinase and tyrosine phosphorylation of the GH receptor itself, as well as Jak2 and the transcription factors Stat1, −3, and −5. In order to study the role of GH receptor tyrosine phosphorylation in intracellular signaling, we constructed GH receptors in which combinations of tyrosines were mutated to phenylalanines. We identified three tyrosine residues at positions 534, 566, and 627 that were required for activation of GH-stimulated transcription of the serine protease inhibitor (Spi) 2.1 promoter. Any of these three tyrosines is able to independently mediate GH-induced transcription, indicating redundancy in this part of the GH receptor. Tyrosine phosphorylation was not required for GH stimulation of mitogen-activated protein (MAP) kinase activity or for GH-stimulated Ca channel activation since these pathways were normal in cells expressing a GH receptor in which all eight intracellular tyrosines were mutated to phenylalanines. Activation of Stat5 by GH was, however, abolished in cells expressing the GH receptor lacking intracellular tyrosines. This study demonstrates that specific tyrosines in the GH receptor are required for transcriptional signaling possibly by their role in the activation of transcription factor Stat5.

Remarkable heterogeneity displayed by oval cells in rat and mouse models of stem cell-mediated liver regeneration.

The experimental protocols used in the investigation of stem cell–mediated liver regeneration in rodents are characterized by activation of the hepatic stem cell compartment in the canals of Hering followed by transit amplification of oval cells and their subsequent differentiation along hepatic lineages. Although the protocols are numerous and often used interchangeably across species, a thorough comparative phenotypic analysis of oval cells in rats and mice using well‐established and generally acknowledged molecular markers has not been provided. In the present study, we evaluated and compared the molecular phenotypes of oval cells in several of the most commonly used protocols of stem cell–mediated liver regeneration—namely, treatment with 2‐acetylaminofluorene and partial (70%) hepatectomy (AAF/PHx); a choline‐deficient, ethionine‐supplemented (CDE) diet; a 3,5‐diethoxycarbonyl‐1,4‐dihydro‐collidin (DDC) diet; and N‐acetyl‐paraaminophen (APAP). Reproducibly, oval cells showing reactivity for cytokeratins (CKs), muscle pyruvate kinase (MPK), the adenosine triphosphate–binding cassette transporter ABCG2/BCRP1 (ABCG2), alpha‐fetoprotein (AFP), and delta‐like protein 1/preadipocyte factor 1 (Dlk/Pref‐1) were induced in rat liver treated according to the AAF/PHx and CDE but not the DDC protocol. In mouse liver, the CDE, DDC, and APAP protocols all induced CKs and ABCG2‐positive oval cells. However, AFP and Dlk/Pref‐1 expression was rarely detected in oval cells. Conclusion: Our results delineate remarkable phenotypic discrepancies exhibited by oval cells in stem cell–mediated liver regeneration between rats and mice and underline the importance of careful extrapolation between individual species. (HEPATOLOGY 2007;45:1462–1470.)

Growth Hormone and Prolactin Stimulate the Expression of Rat Preadipocyte Factor-1/Δ-Like Protein in Pancreatic Islets: Molecular Cloning and Expression Pattern during Development and Growth of the Endocrine Pancreas1

GH and PRL have been shown to stimulate proliferation and insulin production in islets of Langerhans. To identify genes regulated by GH/PRL in islets, we performed differential screening of a complementary DNA library from neonatal rat islets cultured for 24 h with human GH (hGH). One hGH-induced clone had 96% identity with mouse preadipocyte factor-1 (Pref-1, or δ-like protein (Dlk)]. The size of Pref-1 messenger RNA (mRNA) in islets was 1.6 kilobases, with two less abundant mRNAs of 3.7 and 6.2 kilobases. The Pref-1 mRNA content of islets from adult rats was only 1% of that in neonatal islets. Pref-1 mRNA was markedly up-regulated in islets from pregnant rats from day 12 to term compared with those from age-matched female rats. Two peaks in mRNA expression were observed during gestation, one on day 14 and the other at term, whereafter it decreased to nonpregnant levels. Pref-1 mRNA was up-regulated 3- to 4-fold in neonatal rat islets of Langerhans after 48-h culture with hGH, as found also with bovine G...

Interleukin 1 dose-dependently affects the biosynthesis of (pro)insulin in isolated rat islets of Langerhans.

SummaryHuman crude and recombinant interleukin 1 (IL-1) was found to dose- and time-dependently affect the biosynthesis of (pro)insulin in isolated rat islets of Langerhans. Incubation of rat islets with either 0.5 U/ml or 5 U/ml of crude IL-1 for 1 h had no detectable effect on (pro)insulin biosynthesis. After 24 hours of exposure 0.5 U/ml of crude or 0.6 ng/ml of recombinant IL-1 (beta) increased the (pro)insulin biosynthesis by 42% and 58%, respectively, whereas a 10-fold greater concentration of IL-1 decreased the (pro)insulin biosynthesis by 74% and 89%, respectively. The increase in (pro)insulin biosynthesis was accompanied by an increase in total protein biosynthesis indicating a nonspecific stimulatory action of low IL-1 concentrations. In contrast, high IL-1 concentrations caused a more selective decrease of the (pro)insulin biosynthesis when compared to the total protein biosynthesis. In addition, low IL-1 concentrations were found to increase and high concentrations to decrease the relative levels of pre-proinsulin mRNA suggesting that IL-1 may act both at a pre- and post-translational level of insulin biosynthesis.

Cytokine signalling in embryonic stem cells

Cytokines play a central role in maintaining self‐renewal in mouse embryonic stem (ES) cells through a member of the interleukin‐6 type cytokine family termed leukemia inhibitory factor (LIF). LIF activates the JAK‐STAT3 pathway through the class I cytokine receptor gp130, which forms a trimeric complex with LIF and the class I cytokine receptor LIF receptor β. STAT3 has been shown to play a crucial role in self‐renewal in mouse ES cells probably by induction of c‐myc expression. Thus, ablation of STAT3 activation leads to differentiation. However, important connections between STAT3 and other signalling pathways have been documented. In addition, gp130 activation leads to both PI3K and Src activation. The canonical Wnt pathway is sufficient to maintain self‐renewal of both human ES cells and mouse ES cells. It seems quite possible that the main pathway maintaining self‐renewal in ES cells is the Wnt pathway, while the LIF‐JAK‐STAT3 pathway is present in mouse cells as an adaptation for sustaining self‐renewal during embryonic diapause, a condition of delayed implantation in mammals. In keeping with this scenario, the Wnt pathway has been shown to elevate the level of c‐myc. Thus, the two pathways seem to converge on c‐myc as a common target to promote self‐renewal. Whereas LIF does not seem to stimulate self‐renewal in human embryonic stem cells it cannot be excluded that other cytokines are involved. The pleiotropic actions of the increasing number of cytokines and receptors signalling via JAKs, STATs and SOCS exhibit considerable redundancy, compensation and plasticity in stem cells in accordance with the view that stem cells are governed by quantitative variations in strength and duration of signalling events known from other cell types rather than qualitatively different stem cell‐specific factors.