Publications by authors named "Nils Billestrup"

48 Publications

Butyrate Protects Pancreatic Beta Cells from Cytokine-Induced Dysfunction.

Int J Mol Sci 2021 Sep 27;22(19). Epub 2021 Sep 27.

Department of Biomedical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark.

Pancreatic beta cell dysfunction caused by metabolic and inflammatory stress contributes to the development of type 2 diabetes (T2D). Butyrate, produced by the gut microbiota, has shown beneficial effects on glucose metabolism in animals and humans and may directly affect beta cell function, but the mechanisms are poorly described. The aim of this study was to investigate the effect of butyrate on cytokine-induced beta cell dysfunction in vitro. Mouse islets, rat INS-1E, and human EndoC-βH1 beta cells were exposed long-term to non-cytotoxic concentrations of cytokines and/or butyrate to resemble the slow onset of inflammation in T2D. Beta cell function was assessed by glucose-stimulated insulin secretion (GSIS), gene expression by qPCR and RNA-sequencing, and proliferation by incorporation of EdU into newly synthesized DNA. Butyrate protected beta cells from cytokine-induced impairment of GSIS and insulin content in the three beta cell models. Beta cell proliferation was reduced by both cytokines and butyrate. Expressions of the beta cell specific genes , , and reduced by the cytokine IL-1β were not affected by butyrate. In contrast, butyrate upregulated the expression of secretion/transport-related genes and downregulated inflammatory genes induced by IL-1β in mouse islets. In summary, butyrate prevents pro-inflammatory cytokine-induced beta cell dysfunction.
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http://dx.doi.org/10.3390/ijms221910427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8508700PMC
September 2021

Beta-cell dysfunction induced by non-cytotoxic concentrations of Interleukin-1β is associated with changes in expression of beta-cell maturity genes and associated histone modifications.

Mol Cell Endocrinol 2019 10 27;496:110524. Epub 2019 Jul 27.

Department of Biomedical Sciences, University of Copenhagen, Copenhagen, 2200, Denmark; Lead Contact Nils Billestrup, Department of Biomedical Science, University of Copenhagen, Copenhagen, 2200, Denmark. Electronic address:

Decreased insulin secretory capacity in Type 2 diabetes mellitus is associated with beta-cell dedifferentiation and inflammation. We hypothesize that prolonged exposure of beta-cells to low concentrations of IL-1β induce beta-cell dedifferentiation characterized by impaired glucose-stimulated insulin secretion, reduced expression of key beta-cell genes and changes in histone modifications at gene loci known to affect beta-cell function. Ten days exposure to IL-1β at non-cytotoxic concentrations reduced insulin secretion and beta-cell proliferation and decreased expression of key beta-cell identity genes, including MafA and Ucn3 and decreased H3K27ac at the gene loci, suggesting that inflammatory cytokines directly affects the epigenome. Following removal of IL-1β, beta-cell function was normalized and mRNA expression of beta-cell identity genes, such as insulin and Ucn3 returned to pre-stimulation levels. Our findings indicate that prolonged exposure to low concentrations of IL-1β induces epigenetic changes associated with loss of beta-cell identity as observed in Type 2 diabetes.
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http://dx.doi.org/10.1016/j.mce.2019.110524DOI Listing
October 2019

Characterization of the Molecular Mechanisms Underlying Glucose Stimulated Insulin Secretion from Isolated Pancreatic β-cells Using Post-translational Modification Specific Proteomics (PTMomics).

Mol Cell Proteomics 2018 01 7;17(1):95-110. Epub 2017 Nov 7.

From the ‡Department of Biochemistry and Molecular Biology, PR group, University of Southern Denmark, Odense, Denmark;

Normal pancreatic islet β-cells (PBCs) abundantly secrete insulin in response to elevated blood glucose levels, in order to maintain an adequate control of energy balance and glucose homeostasis. However, the molecular mechanisms underlying the insulin secretion are unclear. Improving our understanding of glucose-stimulated insulin secretion (GSIS) mechanisms under normal conditions is a prerequisite for developing better interventions against diabetes. Here, we aimed at identifying novel signaling pathways involved in the initial release of insulin from PBCs after glucose stimulation using quantitative strategies for the assessment of phosphorylated proteins and sialylated -linked (SA) glycoproteins.Islets of Langerhans derived from newborn rats with a subsequent 9-10 days of maturation were stimulated with 20 mm glucose for 0 min (control), 5 min, 10 min, and 15 min. The isolated islets were subjected to time-resolved quantitative phosphoproteomics and sialiomics using iTRAQ-labeling combined with enrichment of phosphorylated peptides and formerly SA glycopeptides and high-accuracy LC-MS/MS. Using bioinformatics we analyzed the functional signaling pathways during GSIS, including well-known insulin secretion pathways. Furthermore, we identified six novel activated signaling pathways ( agrin interactions and prolactin signaling) at 15 min GSIS, which may increase our understanding of the molecular mechanism underlying GSIS. Moreover, we validated some of the regulated phosphosites by parallel reaction monitoring, which resulted in the validation of eleven new phosphosites significantly regulated on GSIS. Besides protein phosphorylation, alteration in SA glycosylation was observed on several surface proteins on brief GSIS. Interestingly, proteins important for cell-cell interaction, cell movement, cell-ECM interaction and Focal Adhesion ( integrins, semaphorins, and plexins) were found regulated at the level of sialylation, but not in protein expression. Collectively, we believe that this comprehensive Proteomics and PTMomics survey of signaling pathways taking place during brief GSIS of primary PBCs is contributing to understanding the complex signaling underlying GSIS.
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http://dx.doi.org/10.1074/mcp.RA117.000217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5750853PMC
January 2018

In-vitro and in-vivo studies supporting the therapeutic potential of ZP3022 in diabetes.

Eur J Pharmacol 2017 Nov 18;815:181-189. Epub 2017 Sep 18.

Department of Biomedical Sciences, University of Copenhagen, Blegdamsvej 3b, 2200 Copenhagen, Denmark.

GLP-1-gastrin dual agonist ZP3022 has been shown to increase β-cell mass with a concomitant improvement of glycemic control in diabetic mice and rats. Here we tested the in-vitro effects of ZP3022 on β-cell proliferation, islet apoptosis and glucose-stimulated insulin secretion (GSIS) in rat islets of Langerhans. Moreover, gene expression profiling in whole pancreas from Zucker Diabetic Fatty (ZDF) rats was performed to characterize genes differently regulated by short-term treatment with ZP3022. Treatments with exendin-4, gastrin-17 alone or in combination were included in the studies. ZP3022 promoted β-cell proliferation, protected from palmitate-, but not from cytokine-induced apoptosis, and induced an increase in GSIS, demonstrating a glucose dependent insulinotropic action of ZP3022 on β-cells. The combination treatment with exendin-4 and gastrin-17 showed comparable effects on proliferation, apoptosis, and GSIS as did ZP3022. Microarray analysis revealed that ZP3022 exerted specific effects on pancreatic gene expression not observed when treating ZDF rats with either exendin-4 alone or in combination with gastrin-17. In particular MAPK signaling pathway was observed among the highest affected pathways; while also pathways related to insulin signaling and secretion were regulated by ZP3022. Moreover, rats treated with ZP3022 had a higher expression of genes encoding for the specific β-cell/endocrine cell markers, such as islet amyloid polypeptide (IAPP), protein convertase 1/3 and -2 (PC 1/3 and-2), as well as transmembrane protein 27(TMEM27) compared to vehicle treated rats. We conclude that ZP3022 may have therapeutic potential in the prevention/delay of β cell dysfunction.
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http://dx.doi.org/10.1016/j.ejphar.2017.09.026DOI Listing
November 2017

Pannexin-2-deficiency sensitizes pancreatic β-cells to cytokine-induced apoptosis in vitro and impairs glucose tolerance in vivo.

Mol Cell Endocrinol 2017 06 5;448:108-121. Epub 2017 Apr 5.

Copenhagen Diabetes Research Center, Pediatric Department, University Hospital Herlev, Denmark. Electronic address:

Pannexins (Panx's) are membrane proteins involved in a variety of biological processes, including cell death signaling and immune functions. The role and functions of Panx's in pancreatic β-cells remain to be clarified. Here, we show Panx1 and Panx2 expression in isolated islets, primary β-cells, and β-cell lines. The expression of Panx2, but not Panx1, was downregulated by interleukin-1β (IL-1β) plus interferon-γ (IFNγ), two pro-inflammatory cytokines suggested to contribute to β-cell demise in type 1 diabetes (T1D). siRNA-mediated knockdown (KD) of Panx2 aggravated cytokine-induced apoptosis in rat INS-1E cells and primary rat β-cells, suggesting anti-apoptotic properties of Panx2. An anti-apoptotic function of Panx2 was confirmed in isolated islets from Panx2 mice and in human EndoC-βH1 cells. Panx2 KD was associated with increased cytokine-induced activation of STAT3 and higher expression of inducible nitric oxide synthase (iNOS). Glucose-stimulated insulin release was impaired in Panx2 islets, and Panx2 mice subjected to multiple low-dose Streptozotocin (MLDS) treatment, a model of T1D, developed more severe diabetes compared to wild type mice. These data suggest that Panx2 is an important regulator of the insulin secretory capacity and apoptosis in pancreatic β-cells.
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http://dx.doi.org/10.1016/j.mce.2017.04.001DOI Listing
June 2017

Regulation of Pancreatic α-Cell Function and Proliferation by Bone Morphogenetic Protein 4 (BMP4) In Vitro.

Endocrinology 2016 Oct 1;157(10):3809-3820. Epub 2016 Aug 1.

Department of Biomedical Sciences, University of Copenhagen, 2200 Copenhagen N, Denmark.

Increased expression of bone morphogenetic proteins (BMPs) in several tissues is associated with inflammation and type 2 diabetes mellitus. BMP2 and BMP4 mRNA expression is increased in pancreatic islets from db/db mice and β-cell proliferation and function are inhibited by BMP4. The effect of BMPs on α-cells is currently unknown. Here, we investigate the effects of BMP4 on mouse and human α-cells in vitro. The effects of BMP4 on α-cell proliferation and function were investigated in islets isolated from male mice and from human donors, and in α-TC1-6 cells. The effects of BMP4 on α-cell function were assessed by determination of glucagon secretion and gene expression. Treatment with BMP4 for 24-96 hours inhibited glucagon secretion in a time-dependent manner in mouse and human islets. Glucagon content, preproglucagon and aristaless related homeobox mRNA expression were reduced after incubation with BMP4 in mouse islets, but not in human islets. The percentage of proliferating α-cells was reduced from 7.3 % to 0.2 % in mouse islets incubated with BMP4. α-cell proliferation in human islets ranged from 0 to 11.8 %, and BMP4 was found to inhibit proliferation of α-cells from all donors when proliferation was present. In agreement with the observations in primary islets, BMP4 decreased glucagon content, preproglucagon, and aristaless related homeobox mRNA expression in α-TC1-6 cells. Our findings suggest that BMP4 has an inhibitory role on glucagon secretion, α-cell growth, and expression of genes maintaining α-cell identity.
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http://dx.doi.org/10.1210/en.2016-1163DOI Listing
October 2016

Aberrant Accumulation of the Diabetes Autoantigen GAD65 in Golgi Membranes in Conditions of ER Stress and Autoimmunity.

Diabetes 2016 09 9;65(9):2686-99. Epub 2016 Jun 9.

Institute of Bioengineering, School of Life Sciences, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Graduate Program in Biotechnology and Bioengineering, School of Life Sciences, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Departments of Medicine, Microbiology and Immunology and Diabetes Center, University of California San Francisco, San Francisco, CA

Pancreatic islet β-cells are particularly susceptible to endoplasmic reticulum (ER) stress, which is implicated in β-cell dysfunction and loss during the pathogenesis of type 1 diabetes (T1D). The peripheral membrane protein GAD65 is an autoantigen in human T1D. GAD65 synthesizes γ-aminobutyric acid, an important autocrine and paracrine signaling molecule and a survival factor in islets. We show that ER stress in primary β-cells perturbs the palmitoylation cycle controlling GAD65 endomembrane distribution, resulting in aberrant accumulation of the palmitoylated form in trans-Golgi membranes. The palmitoylated form has heightened immunogenicity, exhibiting increased uptake by antigen-presenting cells and T-cell stimulation compared with the nonpalmitoylated form. Similar accumulation of GAD65 in Golgi membranes is observed in human β-cells in pancreatic sections from GAD65 autoantibody-positive individuals who have not yet progressed to clinical onset of T1D and from patients with T1D with residual β-cell mass and ongoing T-cell infiltration of islets. We propose that aberrant accumulation of immunogenic GAD65 in Golgi membranes facilitates inappropriate presentation to the immune system after release from stressed and/or damaged β-cells, triggering autoimmunity.
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http://dx.doi.org/10.2337/db16-0180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5001175PMC
September 2016

JNK1 Deficient Insulin-Producing Cells Are Protected against Interleukin-1β-Induced Apoptosis Associated with Abrogated Myc Expression.

J Diabetes Res 2016 10;2016:1312705. Epub 2016 Jan 10.

Immuno-Endocrinology Lab, Endocrinology Research Section, Department of Biomedical Sciences, University of Copenhagen, 2200 Copenhagen N, Denmark; Department of Molecular Medicine and Surgery, Karolinska Institutet, 17177 Stockholm, Sweden.

The relative contributions of the JNK subtypes in inflammatory β-cell failure and apoptosis are unclear. The JNK protein family consists of JNK1, JNK2, and JNK3 subtypes, encompassing many different isoforms. INS-1 cells express JNK1α1, JNK1α2, JNK1β1, JNK1β2, JNK2α1, JNK2α2, JNK3α1, and JNK3α2 mRNA isoform transcripts translating into 46 and 54 kDa isoform JNK proteins. Utilizing Lentiviral mediated expression of shRNAs against JNK1, JNK2, or JNK3 in insulin-producing INS-1 cells, we investigated the role of individual JNK subtypes in IL-1β-induced β-cell apoptosis. JNK1 knockdown prevented IL-1β-induced INS-1 cell apoptosis associated with decreased 46 kDa isoform JNK protein phosphorylation and attenuated Myc expression. Transient knockdown of Myc also prevented IL-1β-induced apoptosis as well as caspase 3 cleavage. JNK2 shRNA potentiated IL-1β-induced apoptosis and caspase 3 cleavage, whereas JNK3 shRNA did not affect IL-1β-induced β-cell death compared to nonsense shRNA expressing INS-1 cells. In conclusion, JNK1 mediates INS-1 cell death associated with increased Myc expression. These findings underline the importance of differentiated targeting of JNK subtypes in the development of inflammatory β-cell failure and destruction.
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http://dx.doi.org/10.1155/2016/1312705DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4745310PMC
December 2016

TRAF2 mediates JNK and STAT3 activation in response to IL-1β and IFNγ and facilitates apoptotic death of insulin-producing β-cells.

Mol Cell Endocrinol 2016 Jan 22;420:24-36. Epub 2015 Nov 22.

Beta-Cell Biology Group, Copenhagen Diabetes Research Center, Department of Paediatrics E, Copenhagen University Hospital Herlev, Herlev, Denmark. Electronic address:

Interleukin-1β (IL-1β) and interferon-γ (IFNγ) contribute to type 1 diabetes (T1D) by inducing β-cell death. Tumor necrosis factor (TNF) receptor-associated factor (TRAF) proteins are adaptors that transduce signaling from a variety of membrane receptors including cytokine receptors. We show here that IL-1β and IFNγ upregulate the expression of TRAF2 in insulin-producing INS-1E cells and isolated rat pancreatic islets. siRNA-mediated knockdown (KD) of TRAF2 in INS-1E cells reduced IL-1β-induced phosphorylation of JNK1/2, but not of p38 or ERK1/2 mitogen-activated protein kinases. TRAF2 KD did not modulate NFκB activation by cytokines, but reduced cytokine-induced inducible nitric oxide synthase (iNOS) promotor activity and expression. We further observed that IFNγ-stimulated phosphorylation of STAT3 required TRAF2. KD of TRAF2 or STAT3 reduced cytokine-induced caspase 3/7 activation, but, intriguingly, potentiated cytokine-mediated loss of plasma membrane integrity and augmented the number of propidium iodide-positive cells. Finally, we found that TRAF2 KD increased cytokine-induced production of reactive oxygen species (ROS). In summary, our data suggest that TRAF2 is an important mediator of IL-1β and IFNγ signaling in pancreatic β-cells.
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http://dx.doi.org/10.1016/j.mce.2015.11.021DOI Listing
January 2016

Inflammatory Cytokines Stimulate Bone Morphogenetic Protein-2 Expression and Release from Pancreatic Beta Cells.

J Interferon Cytokine Res 2016 Jan 26;36(1):20-9. Epub 2015 Aug 26.

1 Section of Cellular and Metabolic Research, Department of Biomedical Sciences, University of Copenhagen , Copenhagen N, Denmark .

The proinflammatory cytokines interleukin-1 beta (IL-1β) and interferon gamma (IFN-γ) play important roles in the progressive loss of beta-cell mass and function during development of both type 1 and type 2 diabetes. We have recently showed that bone morphogenetic protein (BMP)-2 and -4 are expressed in pancreatic islets and inhibit beta-cell growth and function. In this study, we describe that IL-1β and IFN-γ induce the expression of BMP-2 suggesting a possible role for BMP-2 in mediating the effects of IL-1β and IFN-γ on beta-cell apoptosis and dysfunction. IL-1β increased BMP-2 mRNA levels 6- and 3-fold in isolated islets of Langerhans from neonatal rat and human. Downstream target genes of the BMP pathway were also increased by cytokine treatment and could be reversed by neutralization of endogenous BMP activity. Nuclear factor kappa B- (NFκB) binding sites were identified in the rat BMP-2 promoter, and reporter assays verified the role of NFκB in cytokine-induced BMP-2 expression. Electrophoretic mobility shift assay and chromatin immunoprecipitation assays confirmed NFκB binding to BMP-2 promoter upon IL-1β stimulation in beta cells. In conclusion, we suggest that NFκB stimulates BMP-2 mRNA expression in rat and human beta cells upon cytokine exposure.
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http://dx.doi.org/10.1089/jir.2014.0199DOI Listing
January 2016

The anti-diabetic effects of GLP-1-gastrin dual agonist ZP3022 in ZDF rats.

Peptides 2015 Jul 4;69:47-55. Epub 2015 Apr 4.

Research and Development, Zealand Pharma A/S, Glostrup, Denmark.

Aims/hypothesis: Combination treatment with exendin-4 and gastrin has proven beneficial in treatment of diabetes and preservation of beta cell mass in diabetic mice. Here, we examined the chronic effects of a GLP-1-gastrin dual agonist ZP3022 on glycemic control and beta cell dysfunction in overtly diabetic Zucker Diabetic Fatty (ZDF) rats.

Methods: ZDF rats aged 11 weeks were dosed s.c., b.i.d. for 8 weeks with vehicle, ZP3022, liraglutide, exendin-4, or gastrin-17 with or without exendin-4. Glycemic control was assessed by measurements of HbA1c and blood glucose levels, as well as glucose tolerance during an oral glucose tolerance test (OGTT). Beta cell dynamics were examined by morphometric analyses of beta and alpha cell fractions.

Results: ZP3022 improved glycemic control as measured by terminal HbA1c levels (6.2±0.12 (high dose) vs. 7.9±0.07% (vehicle), P<0.001), as did all treatments, except gastrin-17 monotherapy. In contrast, only ZP3022, exendin-4 and combination treatment with exendin-4 and gastrin-17 significantly improved glucose tolerance and increased insulin levels during an OGTT. Moreover, only ZP3022 significantly enhanced the beta cell fraction in ZDF rats, a difference of 41%, when compared to the vehicle group (0.31±0.03 vs. 0.22±0.02%, respectively, P<0.05).

Conclusion: These data suggest that ZP3022 may have therapeutic potential in the prevention/delay of beta cell dysfunction in type 2 diabetes.
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http://dx.doi.org/10.1016/j.peptides.2015.03.024DOI Listing
July 2015

Bone morphogenetic protein 4 inhibits insulin secretion from rodent beta cells through regulation of calbindin1 expression and reduced voltage-dependent calcium currents.

Diabetologia 2015 Jun 2;58(6):1282-90. Epub 2015 Apr 2.

Department of Biomedical Sciences, University of Copenhagen, Nørre Alle 20, 2100, Copenhagen, Denmark.

Aims/hypothesis: Type 2 diabetes is characterised by progressive loss of pancreatic beta cell mass and function. Therefore, it is of therapeutic interest to identify factors with the potential to improve beta cell proliferation and insulin secretion. Bone morphogenetic protein 4 (BMP4) expression is increased in diabetic animals and BMP4 reduces glucose-stimulated insulin secretion (GSIS). Here, we investigate the molecular mechanism behind this inhibition.

Methods: BMP4-mediated inhibition of GSIS was investigated in detail using single cell electrophysiological measurements and live cell Ca(2+) imaging. BMP4-mediated gene expression changes were investigated by microarray profiling, quantitative PCR and western blotting.

Results: Prolonged exposure to BMP4 reduced GSIS from rodent pancreatic islets. This inhibition was associated with decreased exocytosis due to a reduced Ca(2+) current through voltage-dependent Ca(2+) channels. To identify proteins involved in the inhibition of GSIS, we investigated global gene expression changes induced by BMP4 in neonatal rat pancreatic islets. Expression of the Ca(2+)-binding protein calbindin1 was significantly induced by BMP4. Overexpression of calbindin1 in primary islet cells reduced GSIS, and the effect of BMP4 on GSIS was lost in islets from calbindin1 (Calb1) knockout mice.

Conclusions/interpretation: We found BMP4 treatment to markedly inhibit GSIS from rodent pancreatic islets in a calbindin1-dependent manner. Calbindin1 is suggested to mediate the effect of BMP4 by buffering Ca(2+) and decreasing Ca(2+) channel activity, resulting in diminished insulin exocytosis. Both BMP4 and calbindin1 are potential pharmacological targets for the treatment of beta cell dysfunction.
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http://dx.doi.org/10.1007/s00125-015-3568-xDOI Listing
June 2015

Compartmentalization of GABA synthesis by GAD67 differs between pancreatic beta cells and neurons.

PLoS One 2015 3;10(2):e0117130. Epub 2015 Feb 3.

Departments of Medicine and Microbiology/Immunology, Diabetes Center, University of California San Francisco, San Francisco, California, United States of America; Institute of Bioengineering, School of Life Sciences, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.

The inhibitory neurotransmitter GABA is synthesized by the enzyme glutamic acid decarboxylase (GAD) in neurons and in pancreatic β-cells in islets of Langerhans where it functions as a paracrine and autocrine signaling molecule regulating the function of islet endocrine cells. The localization of the two non-allelic isoforms GAD65 and GAD67 to vesicular membranes is important for rapid delivery and accumulation of GABA for regulated secretion. While the membrane anchoring and trafficking of GAD65 are mediated by intrinsic hydrophobic modifications, GAD67 remains hydrophilic, and yet is targeted to vesicular membrane pathways and synaptic clusters in neurons by both a GAD65-dependent and a distinct GAD65-independent mechanism. Herein we have investigated the membrane association and targeting of GAD67 and GAD65 in monolayer cultures of primary rat, human, and mouse islets and in insulinoma cells. GAD65 is primarily detected in Golgi membranes and in peripheral vesicles distinct from insulin vesicles in β-cells. In the absence of GAD65, GAD67 is in contrast primarily cytosolic in β-cells; its co-expression with GAD65 is necessary for targeting to Golgi membranes and vesicular compartments. Thus, the GAD65-independent mechanism for targeting of GAD67 to synaptic vesicles in neurons is not functional in islet β-cells. Therefore, only GAD65:GAD65 homodimers and GAD67:GAD65 heterodimers, but not the GAD67:GAD67 homodimer gain access to vesicular compartments in β-cells to facilitate rapid accumulation of newly synthesized GABA for regulated secretion and fine tuning of GABA-signaling in islets of Langerhans.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0117130PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4315522PMC
January 2016

Surface-expressed insulin receptors as well as IGF-I receptors both contribute to the mitogenic effects of human insulin and its analogues.

J Appl Toxicol 2015 Jul 21;35(7):842-50. Epub 2014 Nov 21.

Centre for Biosecurity and Biopreparedness, Statens Serum Institute, Artillerivej 5, 2300, Copenhagen S, Denmark.

There is a medical need for new insulin analogues. Yet, molecular alterations to the insulin molecule can theoretically result in analogues with carcinogenic effects. Preclinical carcinogenicity risk assessment for insulin analogues rests to a large extent on mitogenicity assays in cell lines. We therefore optimized mitogenicity assay conditions for a panel of five cell lines. All cell lines expressed insulin receptors (IR), IGF-I receptors (IGF-IR) and hybrid receptors, and in all cell lines, insulin as well as the comparator compounds X10 and IGF-I caused phosphorylation of the IR as well as IGF-IR. Insulin exhibited mitogenicity EC(50) values in the single-digit nanomolar to picomolar range. We observed correlations across cell types between (i) mitogenic potency of insulin and IGF-IR/IR ratio, (ii) Akt phosphorylation and mitogenic potency and (iii) Akt phosphorylation and IR phosphorylation. Using siRNA-mediated knockdown of IR and IGF-IR, we observed that in HCT 116 cells the IR appeared dominant in driving the mitogenic response to insulin, whereas in MCF7 cells the IGF-IR appeared dominant in driving the mitogenic response to insulin. Together, our results show that the IR as well as IGF-IR may contribute to the mitogenic potency of insulin. While insulin was a more potent mitogen than IGF-I in cells expressing more IR than IGF-IR, the hyper-mitogenic insulin analogue X10 was a more potent mitogen than insulin across all cell types, supporting that the hyper-mitogenic effect of X10 involves the IR as well as the IGF-IR. These results are relevant for preclinical safety assessment of developmental insulin analogues.
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http://dx.doi.org/10.1002/jat.3082DOI Listing
July 2015

CRFR1 activation protects against cytokine-induced β-cell death.

J Mol Endocrinol 2014 Dec 16;53(3):417-27. Epub 2014 Oct 16.

Clayton Foundation Laboratories for Peptide BiologySalk Institute, 10100 North Torrey Pines Road, La Jolla, California 92037, USACellular and Metabolic Research SectionDepartment of Biomedical Sciences, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen N, Denmark.

During the development of diabetes β-cells are exposed to elevated concentrations of proinflammatory cytokines, TNFα and IL1β, which in vitro induce β-cell death. The class B G-protein-coupled receptors (GPCRs): corticotropin-releasing factor receptor 1 (CRFR1) and CRFR2 are expressed in pancreatic islets. As downstream signaling by other class B GPCRs can protect against cytokine-induced β-cell apoptosis, we evaluated the protective potential of CRFR activation in β-cells in a pro-inflammatory setting. CRFR1/CRFR2 ligands activated AKT and CRFR1 signaling and reduced apoptosis in human islets. In rat and mouse insulin-secreting cell lines (INS-1 and MIN6), CRFR1 agonists upregulated insulin receptor substrate 2 (IRS2) expression, increased AKT activation, counteracted the cytokine-mediated decrease in BAD phosphorylation, and inhibited apoptosis. The anti-apoptotic signaling was dependent on prolonged exposure to corticotropin-releasing factor family peptides and followed PKA-mediated IRS2 upregulation. This indicates that CRFR signaling counteracts proinflammatory cytokine-mediated apoptotic pathways through upregulation of survival signaling in β-cells. Interestingly, CRFR signaling also counteracted basal apoptosis in both cultured INS-1 cells and intact human islets.
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http://dx.doi.org/10.1530/JME-14-0056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4518718PMC
December 2014

Inhibition of beta cell growth and function by bone morphogenetic proteins.

Diabetologia 2014 Dec 27;57(12):2546-54. Epub 2014 Sep 27.

Hagedorn Research Institute, Gentofte, Denmark.

Aims/hypothesis: Impairment of beta cell mass and function is evident in both type 1 and type 2 diabetes. In healthy physiological conditions pancreatic beta cells adapt to the body's increasing insulin requirements by proliferation and improved function. We hypothesised that during the development of diabetes, there is an increase in the expression of inhibitory factors that prevent the beta cells from adapting to the increased need for insulin. We evaluated the effects of bone morphogenetic protein (BMP) 2 and -4 on beta cells.

Methods: The effects of BMP2 and -4 on beta cell proliferation, apoptosis, gene expression and insulin release were studied in isolated islets of Langerhans from rats, mice and humans. The expression of BMPs was analysed by immunocytochemistry and real-time PCR. The role of endogenous BMP was investigated using a soluble and neutralising form of the BMP receptor 1A.

Results: BMP2 and -4 were found to inhibit basal as well as growth factor-stimulated proliferation of primary beta cells from rats and mice. Bmp2 and Bmp4 mRNA and protein were expressed in islets and regulated by inflammatory cytokines. Neutralisation of endogenous BMP activity resulted in enhanced proliferation of rodent beta cells. The expression of Id mRNAs was induced by BMP4 in rat and human islets. Finally, glucose-induced insulin secretion was significantly impaired in rodent and human islets pre-treated with BMP4, and inhibition of BMP activity resulted in enhanced insulin release.

Conclusions/interpretation: These data show that BMP2 and -4 exert inhibitory actions on beta cells in vitro and suggest that BMPs exert regulatory roles of beta cell growth and function.
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http://dx.doi.org/10.1007/s00125-014-3384-8DOI Listing
December 2014

Impact of fetal and neonatal environment on beta cell function and development of diabetes.

Acta Obstet Gynecol Scand 2014 Nov 10;93(11):1109-22. Epub 2014 Oct 10.

Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark; Center for Fetal Programming, Copenhagen, Denmark.

The global epidemic of diabetes is a serious threat against health and healthcare expenses. Although genetics is important it does not explain the dramatic increase in incidence, which must involve environmental factors. Two decades ago the concept of the thrifty phenotype was introduced, stating that the intrauterine environment during pregnancy has an impact on the gene expression that may persist until adulthood and cause metabolic diseases like obesity and type 2 diabetes. As the pancreatic beta cells are crucial in the regulation of metabolism this article will describe the influence of normal pregnancy on the beta cells in both the mother and the fetus and how various conditions like diabetes, obesity, overnutrition and undernutrition during and after pregnancy may influence the ability of the offspring to adapt to changes in insulin demand later in life. The influence of environmental factors including nutrients and gut microbiota on appetite regulation, mitochondrial activity and the immune system that may affect beta cell growth and function directly and indirectly is discussed. The possible role of epigenetic changes in the transgenerational transmission of the adverse programming may be the most threatening aspect with regard to the global diabetes epidemics. Finally, some suggestions for intervention are presented.
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http://dx.doi.org/10.1111/aogs.12504DOI Listing
November 2014

Implications for the offspring of circulating factors involved in beta cell adaptation in pregnancy.

Acta Obstet Gynecol Scand 2014 Nov 16;93(11):1181-9. Epub 2014 Oct 16.

Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark; The Danish Diabetes Academy, Odense University Hospital, Odense, Denmark; Centre for Fetal Programming, Copenhagen, Denmark.

Objective: Several studies have shown an increase in beta cell mass during pregnancy. Somatolactogenic hormones are known to stimulate the proliferation of existing beta cells in rodents whereas the mechanism in humans is still unclear. We hypothesize that in addition to somatolactogenic hormones there are other circulating factors involved in beta cell adaptation to pregnancy. This study aimed at screening for potential pregnancy-associated circulating beta cell growth factors.

Samples: Serum samples from nonpregnant and pregnant women.

Methods: The effect of serum from pregnant women on the proliferation of rat beta cells was studied using [3H]thymidine incorporation and 5-ethynyl-2'-deoxyuridine proliferation assays. In addition, serum from pregnant and nonpregnant women was fractionated by gel filtration and high performance liquid chromatography. The fractionated serum was screened for mitogenic activity in INS-1E cells. Proteins and peptides in mitogenic active serum fractions were identified by amino acid sequencing and mass spectrometry.

Main Outcome Measures: Presence of circulating beta cell proliferating factors.

Results: Late gestational pregnancy serum significantly increased proliferation of rat beta cells compared with early pregnancy and nonpregnancy. The mitogenic active serum fractions contained proteins and peptides derived from kininogen-1, fibrinogen-α, α1-antitrypsin, apolipoprotein-A1, placental lactogen, angiotensinogen and serum albumin.

Conclusion: Pregnancy serum is able to stimulate proliferation of rat beta cells. We have identified several circulating factors that may contribute to beta cell adaptation to pregnancy. Further studies are needed to elucidate their possible role in glucose homeostasis in the mother and her offspring.
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http://dx.doi.org/10.1111/aogs.12505DOI Listing
November 2014

Co-ordinated regulation of neurogenin-3 expression in the maternal and fetal pancreas during pregnancy.

Acta Obstet Gynecol Scand 2014 Nov 23;93(11):1190-7. Epub 2014 Sep 23.

Department of Biomedical Sciences, Cellular and Molecular Medicine Section, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark; Centre for Fetal Programming, Danish Research Council for Strategic Research, Copenhagen, Denmark.

Objective: Several studies have shown increased beta cell mass during pregnancy in both rodents and humans. Proliferation of existing beta cells seems to be the predominant mechanism in rodents, whereas the mechanism in humans is unclear. We hypothesized that neogenesis contributes to the increased beta cell mass in pregnancy and that circulating factors are involved.

Samples: Pancreatic tissue from mice and rat and serum from pregnant women.

Method: Morphometric analysis of pancreas of pregnant and nonpregnant mice was carried out by immunocytochemical staining for the neogenic marker neurogenin-3. Messenger RNA levels of neurogenin-3 and the transcription factor musculoaponeurotic fibrosarcoma oncogene family protein B in fetal rat pancreas cells, cultured with serum from pregnant women, were measured by quantitative polymerase chain reaction.

Main Outcome Measures: The number of neurogenin-3-positive cells present in pregnant mice was increased compared with nonpregnant mice. Neurogenin-3 and musculoaponeurotic fibrosarcoma oncogene family protein B mRNA was detected in fetal rat pancreas exposed to serum from pregnant women.

Results: In pregnant mice we found a 3.6-fold increase in beta cell volume at day 18 compared with nonpregnant mice and a 3.5-fold increase in neurogenin-3 volume at day 14, mainly located in the acinar compartment where it was eightfold higher than in nonpregnant mice. In fetal rat pancreatic cells exposed to serum from pregnant women we found a marked increase in both neurogenin-3 and musculoaponeurotic fibrosarcoma oncogene family protein B mRNA levels in fibroblast-like cells.

Conclusion: These results suggest that neogenesis contributes to the increased beta cell mass in pregnancy and that circulating factors are involved in beta cell formation in both the maternal and fetal pancreas during pregnancy.
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http://dx.doi.org/10.1111/aogs.12495DOI Listing
November 2014

JNK1 protects against glucolipotoxicity-mediated beta-cell apoptosis.

PLoS One 2014 24;9(1):e87067. Epub 2014 Jan 24.

Endocrinology Research Section, Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark ; Department of Molecular Medicine and Surgery, Karolinska Institute, Stockholm, Sweden.

Pancreatic β-cell dysfunction is central to type 2 diabetes pathogenesis. Prolonged elevated levels of circulating free-fatty acids and hyperglycemia, also termed glucolipotoxicity, mediate β-cell dysfunction and apoptosis associated with increased c-Jun N-terminal Kinase (JNK) activity. Endoplasmic reticulum (ER) and oxidative stress are elicited by palmitate and high glucose concentrations further potentiating JNK activity. Our aim was to determine the role of the JNK subtypes JNK1, JNK2 and JNK3 in palmitate and high glucose-induced β-cell apoptosis. We established insulin-producing INS1 cell lines stably expressing JNK subtype specific shRNAs to understand the differential roles of the individual JNK isoforms. JNK activity was increased after 3 h of palmitate and high glucose exposure associated with increased expression of ER and mitochondrial stress markers. JNK1 shRNA expressing INS1 cells showed increased apoptosis and cleaved caspase 9 and 3 compared to non-sense shRNA expressing control INS1 cells when exposed to palmitate and high glucose associated with increased CHOP expression, ROS formation and Puma mRNA expression. JNK2 shRNA expressing INS1 cells did not affect palmitate and high glucose induced apoptosis or ER stress markers, but increased Puma mRNA expression compared to non-sense shRNA expressing INS1 cells. Finally, JNK3 shRNA expressing INS1 cells did not induce apoptosis compared to non-sense shRNA expressing INS1 cells when exposed to palmitate and high glucose but showed increased caspase 9 and 3 cleavage associated with increased DP5 and Puma mRNA expression. These data suggest that JNK1 protects against palmitate and high glucose-induced β-cell apoptosis associated with reduced ER and mitochondrial stress.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0087067PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3901710PMC
February 2015

Lysine deacetylase inhibition prevents diabetes by chromatin-independent immunoregulation and β-cell protection.

Proc Natl Acad Sci U S A 2014 Jan 6;111(3):1055-9. Epub 2014 Jan 6.

Section for Endocrinological Research, Department of Biomedical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark.

Type 1 diabetes is due to destruction of pancreatic β-cells. Lysine deacetylase inhibitors (KDACi) protect β-cells from inflammatory destruction in vitro and are promising immunomodulators. Here we demonstrate that the clinically well-tolerated KDACi vorinostat and givinostat revert diabetes in the nonobese diabetic (NOD) mouse model of type 1 diabetes and counteract inflammatory target cell damage by a mechanism of action consistent with transcription factor--rather than global chromatin--hyperacetylation. Weaning NOD mice received low doses of vorinostat and givinostat in their drinking water until 100-120 d of age. Diabetes incidence was reduced by 38% and 45%, respectively, there was a 15% increase in the percentage of islets without infiltration, and pancreatic insulin content increased by 200%. Vorinostat treatment increased the frequency of functional regulatory T-cell subsets and their transcription factors Gata3 and FoxP3 in parallel to a decrease in inflammatory dendritic cell subsets and their cytokines IL-6, IL-12, and TNF-α. KDACi also inhibited LPS-induced Cox-2 expression in peritoneal macrophages from C57BL/6 and NOD mice. In insulin-producing β-cells, givinostat did not upregulate expression of the anti-inflammatory genes Socs1-3 or sirtuin-1 but reduced levels of IL-1β + IFN-γ-induced proinflammatory Il1a, Il1b, Tnfα, Fas, Cxcl2, and reduced cytokine-induced ERK phosphorylation. Further, NF-κB genomic iNos promoter binding was reduced by 50%, and NF-κB-dependent mRNA expression was blocked. These effects were associated with NF-κB subunit p65 hyperacetylation. Taken together, these data provide a rationale for clinical trials of safety and efficacy of KDACi in patients with autoimmune disease such as type 1 diabetes.
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http://dx.doi.org/10.1073/pnas.1320850111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3903225PMC
January 2014

Divalent metal transporter 1 regulates iron-mediated ROS and pancreatic β cell fate in response to cytokines.

Cell Metab 2012 Oct 20;16(4):449-61. Epub 2012 Sep 20.

Center for Medical Research Methodology, Department of Biomedical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark.

Reactive oxygen species (ROS) contribute to target-cell damage in inflammatory and iron-overload diseases. Little is known about iron transport regulation during inflammatory attack. Through a combination of in vitro and in vivo studies, we show that the proinflammatory cytokine IL-1β induces divalent metal transporter 1 (DMT1) expression correlating with increased β cell iron content and ROS production. Iron chelation and siRNA and genetic knockdown of DMT1 expression reduce cytokine-induced ROS formation and cell death. Glucose-stimulated insulin secretion in the absence of cytokines in Dmt1 knockout islets is defective, highlighting a physiological role of iron and ROS in the regulation of insulin secretion. Dmt1 knockout mice are protected against multiple low-dose streptozotocin and high-fat diet-induced glucose intolerance, models of type 1 and type 2 diabetes, respectively. Thus, β cells become prone to ROS-mediated inflammatory damage via aberrant cellular iron metabolism, a finding with potential general cellular implications.
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http://dx.doi.org/10.1016/j.cmet.2012.09.001DOI Listing
October 2012

Histone deacetylase (HDAC) inhibition as a novel treatment for diabetes mellitus.

Mol Med 2011 May-Jun;17(5-6):378-90. Epub 2011 Jan 25.

Center for Medical Research Methodology, Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark.

Both common forms of diabetes have an inflammatory pathogenesis in which immune and metabolic factors converge on interleukin-1β as a key mediator of insulin resistance and β-cell failure. In addition to improving insulin resistance and preventing β-cell inflammatory damage, there is evidence of genetic association between diabetes and histone deacetylases (HDACs); and HDAC inhibitors (HDACi) promote β-cell development, proliferation, differentiation and function and positively affect late diabetic microvascular complications. Here we review this evidence and propose that there is a strong rationale for preclinical studies and clinical trials with the aim of testing the utility of HDACi as a novel therapy for diabetes.
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http://dx.doi.org/10.2119/molmed.2011.00021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3105132PMC
October 2011

Endothelial progenitor cells in long-standing asymptomatic type 1 diabetic patients with or without diabetic nephropathy.

Nephron Clin Pract 2011 21;118(3):c309-14. Epub 2011 Jan 21.

Steno Diabetes Center, Gentofte, Denmark. hnri @ steno.dk

Unlabelled: A decrease in the number and dysfunction of endothelial progenitor cells (EPC) may increase the risk for progression of cardiovascular disease (CVD) in type 1 diabetic patients with diabetic nephropathy (DN). Our aim was to evaluate EPC numbers in asymptomatic CVD type 1 diabetic patients with or without DN and to study the effect of CVD and medication on EPC numbers.

Methods: We examined EPC numbers in 37 type 1 diabetic patients with DN and 35 type 1 diabetic patients with long-standing normoalbuminuria. Patients were without symptoms of CVD and the prevalence of CVD was previously shown to be very low. EPC number was assessed in in vitro cultures by fluorescent staining of attached cells.

Results: There was no difference in EPC numbers between patients with DN (mean ± SD 120 ± 49 cells/field) and normoalbuminuria (108 ± 41 cells/field; p = 0.25). Furthermore, EPC number was not associated with CVD (p > 0.05). Conventional risk factors were significantly higher in patients with DN and they received more CVD-preventive treatment. All patients receiving simvastatin or calcium-channel blockers had higher numbers of EPC compared to patients not treated with these drugs.

Conclusions: Asymptomatic patients with DN had EPC numbers similar to normoalbuminuric patients, which was related to aggressive CVD intervention therapy. This may have contributed to the low prevalence of CVD.
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http://dx.doi.org/10.1159/000322667DOI Listing
December 2011

Direct demonstration of NCAM cis-dimerization and inhibitory effect of palmitoylation using the BRET2 technique.

FEBS Lett 2011 Jan 26;585(1):58-64. Epub 2010 Nov 26.

Department of Neuroscience and Pharmacology, Institute of Health Sciences, University of Copenhagen, Copenhagen, Denmark.

Biological activity of the neural cell adhesion molecule (NCAM) depends on both adhesion and activation of intra-cellular signaling. Based on in vitro experiments with truncated extra-cellular domains, several models describing homophilic NCAM trans- and cis-interactions have been proposed. However, cis-dimerization in living cells has not been shown directly and the role of the cytoplasmic part in NCAM dimerization is poorly understood. Here, we used the bioluminescence resonance energy transfer (BRET(2)) technique to directly demonstrate that full-length NCAM cis-homodimerizes in living cells. Based on BRET(2)50 values we suggest that the intra-cellular part of NCAM inhibits cis-dimerization, an effect mainly dependent on the palmitoylation sites.
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http://dx.doi.org/10.1016/j.febslet.2010.11.043DOI Listing
January 2011

Deficient SOCS3 and SHP-1 expression in psoriatic T cells.

J Invest Dermatol 2010 Jun 4;130(6):1590-7. Epub 2010 Feb 4.

Department of International Health, Immunology and Microbiology, University of Copenhagen, Blegdamsvej 3c,Copenhagen, Denmark.

IFN-alpha and skin-infiltrating activated T lymphocytes have important roles in the pathogenesis of psoriasis. T cells from psoriatic patients display an increased sensitivity to IFN-alpha, but the pathological mechanisms behind the hyperresponsiveness to IFN-alpha remained unknown. In this study, we show that psoriatic T cells display deficient expression of the suppressor of cytokine signaling (SOCS)3 in response to IFN-alpha and a low baseline expression of the SH2-domain-containing protein-tyrosine phosphatase (SHP)-1 when compared with skin T cells from nonpsoriatic donors. Moreover, IFN-alpha-stimulated psoriatic T cells show enhanced activation of JAKs (JAK1 and TYK2) and signal transducers and activators of transcription. Increased expression of SOCS3 proteins resulting from proteasomal blockade partially inhibits IFN-alpha response. Similarly, forced expression of SOCS3 and SHP-1 inhibits IFN-alpha signaling in psoriatic T cells. In conclusion, our data suggest that loss of regulatory control is involved in the aberrant hypersensitivity of psoriatic T cells to IFN-alpha.
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http://dx.doi.org/10.1038/jid.2010.6DOI Listing
June 2010

CRFR1 is expressed on pancreatic beta cells, promotes beta cell proliferation, and potentiates insulin secretion in a glucose-dependent manner.

Proc Natl Acad Sci U S A 2010 Jan 22;107(2):912-7. Epub 2009 Dec 22.

Peptide Biology Laboratories, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA.

Corticotropin-releasing factor (CRF), originally characterized as the principal neuroregulator of the hypothalamus-pituitary-adrenal axis, has broad central and peripheral distribution and actions. We demonstrate the presence of CRF receptor type 1 (CRFR1) on primary beta cells and show that activation of pancreatic CRFR1 promotes insulin secretion, thus contributing to the restoration of normoglycemic equilibrium. Stimulation of pancreatic CRFR1 initiates a cAMP response that promotes insulin secretion in vitro and in vivo and leads to the phosphorylation of cAMP response element binding and the induction of the expression of several immediate-early genes. Thus, the insulinotropic actions of pancreatic CRFR1 oppose the activation of CRFR1 on anterior pituitary corticotropes, leading to the release of glucocorticoids that functionally antagonize the actions of insulin. Stimulation of the MIN6 insulinoma line and primary rat islets with CRF also activates the MAPK signaling cascade leading to rapid phosphorylation of Erk1/2 in response to CRFR1-selective ligands, which induce proliferation in primary rat neonatal beta cells. Importantly, CRFR1 stimulates insulin secretion only during conditions of intermediate to high ambient glucose, and the CRFR1-dependent phosphorylation of Erk1/2 is greater with elevated glucose concentrations. This response is reminiscent of the actions of the incretins, which potentiate insulin secretion only during elevated glucose conditions. The presence of CRFR1 on beta cells adds another layer of complexity to the intricate network of paracrine and autocrine factors and their cognate receptors whose coordinated efforts can dictate islet hormone output and regulate beta cell proliferation.
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http://dx.doi.org/10.1073/pnas.0913610107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2818901PMC
January 2010

Dissociation between skeletal muscle inhibitor-kappaB kinase/nuclear factor-kappaB pathway activity and insulin sensitivity in nondiabetic twins.

J Clin Endocrinol Metab 2010 Jan 29;95(1):414-21. Epub 2009 Oct 29.

Steno Diabetes Center, DK-2820 Gentofte, Denmark.

Context: Several studies suggest a link between increased activity of the inflammatory inhibitor-kappaB kinase/nuclear factor-kappaB (IKK/NF-kappaB) pathway in skeletal muscle and insulin resistance.

Objective: We aimed to study the regulation of skeletal muscle IKK/NF-kappaB pathway activity as well as the association with glucose metabolism and skeletal muscle insulin signaling.

Methods: The study population included a metabolically well-characterized cohort of young and elderly predominantly nondiabetic twins (n = 181). Inhibitor-kappaBbeta (IkappaBbeta) protein levels are negatively associated with IKK/NF-kappaB pathway activity and were used to evaluate pathway activity with p65 levels included as loading control. This indirect measure for IKK/NF-kappaB pathway activity was validated by a p65 binding assay.

Results: Evaluating the effects of heritability, age, sex, obesity, aerobic capacity, and several hormonal factors (eg insulin and TNF-alpha), only sex and age were significant predictors of IkappaBbeta to p65 ratio (28% decreased ratio in the elderly, P < 0.01, and 49% increased in males P < 0.01). IkappaBbeta to p65 ratio was unrelated to peripheral insulin sensitivity (P = 0.51) and in accordance with this also unrelated to proximal insulin signaling (P = 0.81). Although no association was seen with plasma glucose after oral glucose challenge, there was a tendency for lower IkappaBbeta to p65 ratio (adjusted for age and sex) in subjects with impaired as opposed to normal glucose tolerance (P = 0.055).

Conclusions: Altogether the subtle elevated IKK/NF-kappaB pathway activity seen in glucose-intolerant subjects suggests that IKK/NF-kappaB pathway activation may be secondary to impaired glucose tolerance and that skeletal muscle IKK/NF-kappaB pathway activity is unlikely to play any major role in the control of skeletal muscle insulin action in nondiabetic subjects.
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http://dx.doi.org/10.1210/jc.2009-1147DOI Listing
January 2010

The ectopic expression of Pax4 in the mouse pancreas converts progenitor cells into alpha and subsequently beta cells.

Cell 2009 Aug;138(3):449-62

Department of Molecular Cell Biology, Max-Planck Institute for Biophysical Chemistry, Am Fassberg, D-37077 Göttingen, Germany.

We have previously reported that the loss of Arx and/or Pax4 gene activity leads to a shift in the fate of the different endocrine cell subtypes in the mouse pancreas, without affecting the total endocrine cell numbers. Here, we conditionally and ectopically express Pax4 using different cell-specific promoters and demonstrate that Pax4 forces endocrine precursor cells, as well as mature alpha cells, to adopt a beta cell destiny. This results in a glucagon deficiency that provokes a compensatory and continuous glucagon+ cell neogenesis requiring the re-expression of the proendocrine gene Ngn3. However, the newly formed alpha cells fail to correct the hypoglucagonemia since they subsequently acquire a beta cell phenotype upon Pax4 ectopic expression. Notably, this cycle of neogenesis and redifferentiation caused by ectopic expression of Pax4 in alpha cells is capable of restoring a functional beta cell mass and curing diabetes in animals that have been chemically depleted of beta cells.
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http://dx.doi.org/10.1016/j.cell.2009.05.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2792203PMC
August 2009
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