Rikard G. Fred

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.

Imatinib mesilate-induced phosphatidylinositol 3-kinase signalling and improved survival in insulin-producing cells: role of Src homology 2-containing inositol 5′-phosphatase interaction with c-Abl

Aims/hypothesisIt is not clear how small tyrosine kinase inhibitors, such as imatinib mesilate, protect against diabetes and beta cell death. The aim of this study was to determine whether imatinib, as compared with the non-cAbl-inhibitor sunitinib, affects pro-survival signalling events in the phosphatidylinositol 3-kinase (PI3K) pathway.MethodsHuman EndoC-βH1 cells, murine beta TC-6 cells and human pancreatic islets were used for immunoblot analysis of insulin receptor substrate (IRS)-1, Akt and extracellular signal-regulated kinase (ERK) phosphorylation. Phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] plasma membrane concentrations were assessed in EndoC-βH1 and MIN6 cells using evanescent wave microscopy. Src homology 2-containing inositol 5′-phosphatase 2 (SHIP2) tyrosine phosphorylation and phosphatase and tensin homologue deleted on chromosome 10 (PTEN) serine phosphorylation, as well as c-Abl co-localisation with SHIP2, were studied in HEK293 and EndoC-βH1 cells by immunoprecipitation and immunoblot analysis. Gene expression was assessed using RT-PCR. Cell viability was measured using vital staining.ResultsImatinib stimulated ERK(thr202/tyr204) phosphorylation in a c-Abl-dependent manner. Imatinib, but not sunitinib, also stimulated IRS-1(tyr612), Akt(ser473) and Akt(thr308) phosphorylation. This effect was paralleled by oscillatory bursts in plasma membrane PI(3,4,5)P3 levels. Wortmannin induced a decrease in PI(3,4,5)P3 levels, which was slower in imatinib-treated cells than in control cells, indicating an effect on PI(3,4,5)P3-degrading enzymes. In line with this, imatinib decreased the phosphorylation of SHIP2 but not of PTEN. c-Abl co-immunoprecipitated with SHIP2 and its binding to SHIP2 was largely reduced by imatinib but not by sunitinib. Imatinib increased total β-catenin levels and cell viability, whereas sunitinib exerted negative effects on cell viability.Conclusions/interpretationImatinib inhibition of c-Abl in beta cells decreases SHIP2 activity, which results in enhanced signalling downstream of PI3 kinase.

Receptor for advanced glycation end products (RAGE) knockout reduces fetal dysmorphogenesis in murine diabetic pregnancy

The receptor for Advanced Glycation End products (RAGE) is implicated in the pathogenesis of diabetic complications, but its importance in diabetic embryopathy is unclear. We therefore investigated the role of RAGE in diabetic embryopathy using streptozotocin induced diabetes in female wild type (WT) C57Bl/6N and RAGE knockout C57Bl/6N (RAGE(-/-)) mice, mated with control males of the same genotype. Maternal diabetes induced more fetal resorption and malformation (facial skeleton, neural tube) in the WT than in the RAGE(-/-) fetuses. Maternal plasma glucose and methylgyoxal concentrations, as well as embryonic N(ε)-carboxymethyl-lysine (CML) levels were increased to the same extent in diabetic WT and RAGE(-/-) pregnancy. However, maternal diabetes induced increased fetal hepatic isoprostane 8-iso-PGF2α levels (oxidative stress marker) and embryonic activation of NFκB in WT only (not in RAGE(-/-) embryos). The association between RAGE knockout and diminished embryonic dysmorphogenesis in diabetic pregnancy suggests that embryonic RAGE activation is involved in diabetic embryopathy.

The H1-receptor antagonist cetirizine protects partially against cytokine- and hydrogen peroxide-induced β-TC6 cell death in vitro.

Objective It has been proposed that the histamine 1 (H1) receptor not only promotes allergic reactions but also modulates autoimmune diseases, such as type 1 diabetes. In line with this, it has recently been reported that the H1-receptor antagonist cetirizine can counteract the activation of signals/factors pertinent to the pathogenesis of type 1 diabetes and cytokine-induced &bgr;-cell destruction. Therefore, the overall aim of this study was to determine whether H1-receptor antagonists affect cytokine-induced &bgr;-cell death and signaling in vitro. Methods The insulin-producing cell line &bgr;-TC6 was exposed to the proinflammatory cytokines interleukin 1&bgr;+ interferon &ggr;, or hydrogen peroxide. The H1-receptor antagonists desloratadine and cetirizine were added to the cell cultures and cell viability; macrophage inhibitory factor levels, c-Jun N-terminal kinase phosphorylation, c-Jun expression, and &bgr;-catenin levels were analyzed by flow cytometry, real-time polymerase chain reaction, and immunoblotting. Results Cetirizine protected partially against both cytokine- and hydrogen peroxide–induced cell death. This effect was paralleled by an inhibition of cytokine-induced c-Jun N-terminal kinase phosphorylation, c-Jun induction, and a restoration of macrophage inhibitory factor contents. Cetirizine also increased the &bgr;-TC6 cell contents of &bgr;-catenin at basal conditions. Conclusions Our results indicate a protective effect of a specific H1-receptor antagonist.

Addition of exogenous sodium palmitate increases the IAPP/insulin mRNA ratio via GPR40 in human EndoC-beta H1 cells

Abstract Background: Enhanced IAPP production may contribute to islet amyloid formation in type 2 diabetes. The objective of this study was to determine the effects of the saturated fatty acid palmitate on IAPP levels in human β-cells. Methods: EndoC-βH1 cells and human islets were cultured in the presence of sodium palmitate. Effects on IAPP/insulin mRNA expression and secretion were determined using real-time qPCR/ELISA. Pharmacological activators and/or inhibitors and RNAi were used to determine the underlying mechanisms. Results: We observed that EndoC-βH1 cells exposed to palmitate for 72 h displayed decreased expression of Pdx-1 and MafA and increased expression of thioredoxin-interacting protein (TXNIP), reduced insulin mRNA expression and glucose-induced insulin secretion, as well as increased IAPP mRNA expression and secretion. Further, these effects were independent of fatty acid oxidation, but abolished in response to GPR40 inhibition/downregulation. In human islets both a high glucose concentration and palmitate promoted increased IAPP mRNA levels, resulting in an augmented IAPP/insulin mRNA ratio. This was paralleled by elevated IAPP/insulin protein secretion and content ratios. Conclusions: Addition of exogenous palmitate to human β-cells increased the IAPP/insulin expression ratio, an effect contributed to by activation of GPR40. These findings may be pertinent to our understanding of the islet amyloid formation process.

Co-culture of insulin producing human EndoC-βH1 cells with boundary cap neural crest stem cells protects partially against cytokine-induced cell death

We have recently observed that co-culture of mouse and rat beta-cells with mouse boundary cap neural crest stem cells (bNCSCs) protected against inflammatory cytokine-induced beta-cell death, possibly via direct cadherinmediated cell-to-cell junctions. However, it has not been addressed whether also human beta-cells can be protected via this strategy. If possible it would be an important approach for development of new protocols for improved outcome of islet transplantation to Type-1 diabetes patients. The aim of this investigation was therefore to study the effect of bNCSC co-culture with insulin producing human EndoC-βH1 cells on cytokine-induced cell death. For this purpose GFP-positive bNCSCs were cultured together with GFP-negative human EndoC-βH1 cells in the presence of the cytokines IL-1γ (50 U/ml) and IFN-γ (1000 U/ml). Cells were then stained with propidium iodide and trypsinized for flow cytometry analysis. Analysis of propidium iodide fluorescence in GFP-positive and GFP-negative cells revealed that EndoC-βH1 cells died to a lower extent when co-cultured with bNCSCs than when cultured without bNCSCs. We also observed that EndoC-βH1 cells formed N-cadherin, but not E-cadherin junctions with the bNCSCs. The bNCSC cell population contained a large proportion of beta-tubulin expressing cells indicating neuronal differentiation. A protective function of the N-cadherin junctions was verified by the finding that a neutralizing N-cadherin antibody counteracted the effect of co-culture. We conclude that the interaction between human insulin producing cells and bNCSCs results in a lowered susceptibility of insulin producing cells to pro-inflammatory cytokines in vitro.

PTB and TIAR binding to insulin mRNA 3'- and 5'UTRs; implications for insulin biosynthesis and messenger stability

Objectives Insulin expression is highly controlled on the posttranscriptional level. The RNA binding proteins (RBPs) responsible for this result are still largely unknown. Methods and results To identify RBPs that bind to insulin mRNA we performed mass spectrometry analysis on proteins that bound synthetic oligonucloetides mimicing the 5′- and the 3′-untranslated regions (UTRs) of rat and human insulin mRNA in vitro. We observed that the RBPs heterogeneous nuclear ribonucleoprotein (hnRNP) U, polypyrimidine tract binding protein (PTB), hnRNP L and T-cell restricted intracellular antigen 1-related protein (TIA-1-related protein; TIAR) bind to insulin mRNA sequences, and that the in vitro binding affinity of these RBPs changed when INS-1 cells were exposed to glucose, 3-isobutyl-1-methylxanthine (IBMX) or nitric oxide. High glucose exposure resulted in a modest increase in PTB and TIAR binding to an insulin mRNA sequence. The inducer of nitrosative stress DETAnonoate increased markedly hnRNP U and TIAR mRNA binding. An increased PTB to TIAR binding ratio in vitro correlated with higher insulin mRNA levels and insulin biosynthesis rates in INS-1 cells. To further investigate the importance of RNA-binding proteins for insulin mRNA stability, we decreased INS-1 and EndoC-βH1 cell levels of PTB and TIAR by RNAi. In both cell lines, decreased levels of PTB resulted in lowered insulin mRNA levels while decreased levels of TIAR resulted in increased insulin mRNA levels. Thapsigargin-induced stress granule formation was associated with a redistribution of TIAR from the cytosol to stress granules. Conclusions These experiments indicate that alterations in insulin mRNA stability and translation correlate with differential RBP binding. We propose that the balance between PTB on one hand and TIAR on the other participates in the control of insulin mRNA stability and utilization for insulin biosynthesis.