Lars Groth Grunnet

Proinflammatory Cytokines Activate the Intrinsic Apoptotic Pathway in β-Cells

OBJECTIVE Proinflammatory cytokines are cytotoxic to β-cells and have been implicated in the pathogenesis of type 1 diabetes and islet graft failure. The importance of the intrinsic mitochondrial apoptotic pathway in cytokine-induced β-cell death is unclear. Here, cytokine activation of the intrinsic apoptotic pathway and the role of the two proapoptotic Bcl-2 proteins, Bad and Bax, were examined in β-cells. RESEARCH DESIGN AND METHODS Human and rat islets and INS-1 cells were exposed to a combination of proinflammatory cytokines (interleukin-1β, interferon-γ, and/or tumor necrosis factor-α). Activation of Bad was determined by Ser136 dephosphorylation, mitochondrial stress by changes in mitochondrial metabolic activity and cytochrome c release, downstream apoptotic signaling by activation of caspase-9 and -3, and DNA fragmentation. The inhibitors FK506 and V5 were used to investigate the role of Bad and Bax activation, respectively. RESULTS We found that proinflammatory cytokines induced calcineurin-dependent dephosphorylation of Bad Ser136, mitochondrial stress, cytochrome c release, activation of caspase-9 and -3, and DNA fragmentation. Inhibition of Bad Ser136 dephosphorylation or Bax was found to inhibit cytokine-induced intrinsic proapoptotic signaling. CONCLUSIONS Our findings demonstrate that the intrinsic mitochondrial apoptotic pathway contributes significantly to cytokine-induced β-cell death and suggest a functional role of calcineurin-mediated Bad Ser136 dephosphorylation and Bax activity in cytokine-induced apoptosis.

High glucose suppresses human islet insulin biosynthesis by inducing miR-133a leading to decreased polypyrimidine tract binding protein-expression.

Background Prolonged periods of high glucose exposure results in human islet dysfunction in vitro. The underlying mechanisms behind this effect of high glucose are, however, unknown. The polypyrimidine tract binding protein (PTB) is required for stabilization of insulin mRNA and the PTB mRNA 3′-UTR contains binding sites for the microRNA molecules miR-133a, miR-124a and miR-146. The aim of this study was therefore to investigate whether high glucose increased the levels of these three miRNAs in association with lower PTB levels and lower insulin biosynthesis rates. Methodology/Principal Findings Human islets were cultured for 24 hours in the presence of low (5.6 mM) or high glucose (20 mM). Islets were also exposed to sodium palmitate or the proinflammatory cytokines IL-1β and IFN-γ, since saturated free fatty acids and cytokines also cause islet dysfunction. RNA was then isolated for real-time RT-PCR analysis of miR-133a, miR-124a, miR-146, insulin mRNA and PTB mRNA contents. Insulin biosynthesis rates were determined by radioactive labeling and immunoprecipitation. Synthetic miR-133a precursor and inhibitor were delivered to dispersed islet cells by lipofection, and PTB was analyzed by immunoblotting following culture at low or high glucose. Culture in high glucose resulted in increased islet contents of miR-133a and reduced contents of miR-146. Cytokines increased the contents of miR-146. The insulin and PTB mRNA contents were unaffected by high glucose. However, both PTB protein levels and insulin biosynthesis rates were decreased in response to high glucose. The miR-133a inhibitor prevented the high glucose-induced decrease in PTB and insulin biosynthesis, and the miR-133a precursor decreased PTB levels and insulin biosynthesis similarly to high glucose. Conclusion Prolonged high-glucose exposure down-regulates PTB levels and insulin biosynthesis rates in human islets by increasing miR-133a levels. We propose that this mechanism contributes to hyperglycemia-induced beta-cell dysfunction.

Lysine deacetylases are produced in pancreatic beta cells and are differentially regulated by proinflammatory cytokines

Aims/hypothesisCytokine-induced beta cell toxicity is abrogated by non-selective inhibitors of lysine deacetylases (KDACs). The KDAC family consists of 11 members, namely histone deacetylases HDAC1 to HDAC11, but it is not known which KDAC members play a role in cytokine-mediated beta cell death. The aim of the present study was to examine the KDAC gene expression profile of the beta cell and to investigate whether KDAC expression is regulated by cytokines. In addition, the protective effect of the non-selective KDAC inhibitor ITF2357 and interdependent regulation of four selected KDACs were investigated.MethodsThe beta cell line INS-1 and intact rat and human islets were exposed to cytokines with or without ITF2357. Expression of mRNA was assessed by real-time PCR and selected targets validated at the protein level by immunoblotting. Effects on cytokine-induced toxicity were investigated by in vitro assays.ResultsHdac1 to Hdac11 were expressed and differentially regulated by cytokines in INS-1 cells and rat islets. HDAC1, -2, -6 and -11 were found to be expressed and regulated by cytokines in human islets. ITF2357 protected against cytokine-induced beta cell apoptosis and counteracted cytokine-induced attenuation of basal insulin secretion. In addition, cytokine-induced regulation of Hdac2 and Hdac6, but not Hdac1 and Hdac11, was reduced by KDAC inhibition.Conclusions/interpretationAll classical KDAC genes are expressed by beta cells and differentially regulated by cytokines. Based on the relative expression levels and degree of regulation by cytokines, we propose that HDAC1, -2, -6 and -11 are of particular importance for beta cell function. These observations may help in the design of specific KDAC inhibitors to prevent beta cell destruction in situ and in islet grafts.

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

Significance Type 1 diabetes is due to immune-mediated pancreatic β-cell destruction. Lysine deacetylase inhibitors (KDACi) protect β-cells from inflammatory destruction in vitro and are promising immunomodulators. The orally active and clinically well-tolerated KDACi vorinostat and givinostat reverted diabetes in a mouse model of type 1 diabetes and counteracted inflammatory target cell damage. Importantly, these effects were achieved with doses that are safe and effective in human inflammatory diseases. Of note, the mechanism of action was compatible with transcription factor—rather than global chromatin—hyperacetylation, causing inhibition of transcription factor binding and reduction of proinflammatory gene expression in leukocytes and β-cells. These data provide a rationale for clinical trials of safety and efficacy of KDACi in patients with Type 1 diabetes. 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.

Inhibition of Nuclear Factor-κB or Bax Prevents Endoplasmic Reticulum Stress- But Not Nitric Oxide-Mediated Apoptosis in INS-1E Cells

Accumulating evidence suggests that endoplasmic reticulum (ER) stress by mechanisms that include ER Ca(2+) depletion via NO-dependent down-regulation of sarcoendoplasmic reticulum Ca(2+) ATPase 2b (SERCA2b) contributes to beta-cell death in type 1 diabetes. To clarify whether the molecular pathways elicited by NO and ER Ca(2+) depletion differ, we here compare the direct effects of NO, in the form of the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP), with the effects of SERCA2 inhibitor thapsigargin (TG) on MAPK, nuclear factor kappaB (NFkappaB), Bcl-2 proteins, ER stress, and apoptosis. Exposure of INS-1E cells to TG or SNAP caused caspase-3 cleavage and apoptosis. Both TG and SNAP induced activation of the proapoptotic transcription factor CCAAT/enhancer-binding protein homologous protein (CHOP). However, other classical ER stress-induced markers such as up-regulation of ER chaperone Bip and alternative splicing of the transcription factor Xbp-1 were exclusively activated by TG. TG exposure caused NFkappaB activation, as assessed by IkappaB degradation and NFkappaB DNA binding. Inhibition of NFkappaB or the Bcl-2 family member Bax pathways protected beta-cells against TG- but not SNAP-induced beta-cell death. These data suggest that NO generation and direct SERCA2 inhibition cause two quantitative and qualitative different forms of ER stress. In contrast to NO, direct ER stress induced by SERCA inhibition causes activation of ER stress signaling pathways and elicit proapoptotic signaling via NFkappaB and Bax.

Transporter function and cyclic AMP turnover in normal colonic mucosa from patients with and without colorectal neoplasia

BackgroundThe pathogenesis of colorectal neoplasia is still unresolved but has been associated with alterations in epithelial clearance of xenobiotics and metabolic waste products. The aim of this study was to functionally characterize the transport of cyclic nucleotides in colonic biopsies from patients with and without colorectal neoplasia.MethodsCyclic nucleotides were used as model substrates shared by some OATP- and ABC-transporters, which in part are responsible for clearance of metabolites and xenobiotics from the colonic epithelium. On colonic biopsies from patients with and without colorectal neoplasia, molecular transport was electrophysiologically registered in Ussing-chamber set-ups, mRNA level of selected transporters was quantified by rt-PCR, and subcellular location of transporters was determined by immunohistochemistry.ResultsOf four cyclic nucleotides, dibuturyl-cAMP induced the largest short circuit current in both patient groups. The induced short circuit current was significantly lower in neoplasia-patients (p = 0.024). The observed altered transport of dibuturyl-cAMP in neoplasia-patients could not be directly translated to an observed increased mRNA expression of OATP4A1 and OATP2B1 in neoplasia patients. All other examined transporters were expressed to similar extents in both patient groups.ConclusionsOATP1C1, OATP4A1, OATP4C1 seem to be involved in the excretory system of human colon. ABCC4 is likely to be involved from an endoplasmic-Golgi complex and basolateral location in goblet cells. ABCC5 might be directly involved in the turnover of intracellular cAMP at the basolateral membrane of columnar epithelial cells, while OATP2B1 is indirectly related to the excretory system. Colorectal neoplasia is associated with lower transport or sensitivity to cyclic nucleotides and increased expression of OATP2B1 and OATP4A1 transporters, known to transport PGE2.

Macrophage contact dependent and independent TLR4 mechanisms induce β-cell dysfunction and apoptosis in a mouse model of type 2 diabetes.

Type 2 diabetes (T2D) is evolving into a global disease and patients have a systemic low-grade inflammation, yet the role of this inflammation is still not established. One plausible mechanism is enhanced expression and activity of the innate immune system. Therefore, we evaluated the expression and the function of the toll-like receptor 4 (TLR4) on pancreatic β-cells in primary mouse islets and on the murine β-cell line MIN6 in the presence or absence of macrophages. Diabetic islets have 40% fewer TLR4 positive β-cells, but twice the number of TLR4 positive macrophages as compared to healthy islets. Healthy and diabetic islets respond to a TLR4 challenge with enhanced production of cytokines (5–10-fold), while the TLR4 negative β-cell line MIN6 fails to produce cytokines. TLR4 stimulation induces β-cell dysfunction in mouse islets, measured as reduced glucose stimulated insulin secretion. Diabetic macrophages from 4-months old mice have acquired a transient enhanced capacity to produce cytokines when stimulated with LPS. Interestingly, this is lost in 6-months old diabetic mice. TLR4 activation alone does not induce apoptosis in islets or MIN-6 cells. In contrast, macrophages mediate TLR4-dependent cell-contact dependent (3-fold) as well as cell-contact independent (2-fold) apoptosis of both islets and MIN-6 cells. Importantly, diabetic macrophages have a significantly enhanced capacity to induce β-cell apoptosis compared to healthy macrophages. Taken together, the TLR4 responsiveness is elevated in the diabetic islets and mainly mediated by newly recruited macrophages. The TLR4 positive macrophages, in both a cell-contact dependent and independent manner, induce apoptosis of β-cells in a TLR4 dependent fashion and TLR4 activation directly induces β-cell dysfunction. Thus, targeting either the TLR4 pathway or the macrophages provides a novel attractive treatment regime for T2D.

Altering β-cell number through stable alteration of miR-21 and miR-34a expression

Aim: An insufficient functional β-cell mass is a prerequisite to develop diabetes. Thus, means to protect or restore β-cell mass are important goals in diabetes research. Inflammation and proinflammatory cytokines play important roles in β-cell dysfunction and death, and recent data show that 2 miRNAs, miR-21 and miR-34a, may be involved in mediating cytokine-induced β-cell dysfunction. Therefore, manipulation of miR-21 and miR-34a levels may potentially be beneficial to β cells. To study the effect of long-term alterations of miR-21 or miR-34a levels upon net β-cell number, we stably overexpressed miR-21 and knocked down miR-34a, and investigated essential cellular processes. Materials and Methods: miRNA expression was manipulated using Lentiviral transduction of the β-cell line INS-1. Stable cell lines were generated, and cell death, NO synthesis, proliferation, and total cell number were monitored in the absence or presence of cytokines. Results: Overexpression of miR-21 decreased net β-cell number in the absence of cytokines, and increased apoptosis and NO synthesis in the absence and presence of cytokines. Proliferation was increased upon miR-21 overexpression. Knockdown of miR-34a increased net β-cell number in the absence of cytokines, and reduced apoptosis and NO synthesis in the absence and presence of cytokines. Proliferation was decreased upon miR-34a knockdown. Conclusion: As overexpression of miR-21 increased proliferation, but also apoptosis and NO synthesis, the potential of miR-21 as a therapeutic agent to increase β-cell survival is doubtful. Knockdown of miR-34a slightly decreased proliferation, but as apoptosis and NO synthesis were highly reduced, miR-34a may be further investigated as a therapeutic target to reduce β-cell death and dysfunction.

Cytokines and Type 1 Diabetes: A Numbers Game

Cytokines are protein mediators of intercellular stress communication with autocrine, paracrine, and endocrine modes of action. Hormones differ from cytokines by being produced in specialized glandular organs, by not all being proteins, and by their homeostatic properties. Virtually all nucleated cells produce and respond to cytokines under defined conditions, mainly in response to stress signals and in parallel to their differentiated functions. The cytokine group of molecules encompasses several hundred individual protein moieties, including 37 interleukins (ILs) and a multitude of chemokines. Many of these molecules have redundant functions whereas others elicit specific cellular reactions. It is debated whether cytokines also have homeostatic properties, i.e., contribute to the maintenance of normal cellular physiology. Cytokines are central mediators of inflammation by controling innate and adaptive immune responses as well as tissue damage, defense, repair, and remodeling. Type 1 diabetes is an inflammatory disease of the pancreatic islet, in which insulin-producing β-cells are preferentially destroyed to varying degrees by the concerted action of autoreactive T-cells and monocytic cells (1). Despite huge research efforts over the past 40 years, no therapy targeting pathogenetic events in type 1 diabetes has attained routine clinical utility. A number of cytokines have been shown to be important for the development of type 1 diabetes both at the level of the immune system and at the level of the target β-cells (2,3). The actual mechanism of β-cell destruction is still unclear, and classical T-cell effector pathways as well as many proinflammatory cytokines have been proven dispensable in transgenic animal models. Only a few …

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

MINT‐8071483: NCAM140 (uniprotkb:P13591‐1) physically interacts (MI:0915) with NCAM140 (uniprotkb:P13591‐1) by competition binding (MI:0405)