Visual impairment and mortality in patients with type 2 diabetes.

Objective: To evaluate whether visual acuity impairment was an independent predictor of mortality in patients with type 2 diabetes. Research design and methods: This is a 19-year follow-up of a cohort of 1241 patients newly diagnosed with type 2 diabetes and aged 40 years or over. Visual acuity was assessed by practicing ophthalmologists both at diabetes diagnosis and after 6 years. The logarithmic value of the visual acuity (logMAR) was the exposure. Multivariable Cox regression models were adjusted for multiple potential confounders including cardiovascular disease, and censored for potential mediators, that is, fractures/trauma. Primary outcomes were from national registers: all-cause mortality and diabetes-related mortality. Results: Visual impairment at diabetes diagnosis was robustly associated with subsequent 6-year all-cause mortality. Per 1 unit reduced logMAR acuity the incidence rate of all-cause mortality increased with 51% (adjusted HR: 1.51; 95% CI 1.12 to 2.03) and of fractures/trauma with 59% (HR: 1.59; 95% CI 1.18 to 2.15), but visual acuity was not associated with diabetes-related mortality. After censoring for fractures/trauma, visual acuity was still an independent risk factor for all-cause mortality (HR: 1.68; 95% CI 1.23 to 2.30). In contrast, visual acuity 6 years after diabetes diagnosis was not associated with the subsequent 13 years' incidence of any of the outcomes, as an apparent association with all-cause mortality and diabetes-related mortality was explained by confounding from comorbidity. Conclusions: Visual acuity measured by ophthalmologists in patients newly diagnosed with type 2 diabetes was an independent predictor of mortality in the short term.

MicroRNAs and histone deacetylase inhibition-mediated protection against inflammatory ß-cell damage.

Inflammatory ß-cell failure contributes to type 1 and type 2 diabetes pathogenesis. Pro-inflammatory cytokines cause ß-cell dysfunction and apoptosis, and lysine deacetylase inhibitors (KDACi) prevent ß-cell failure in vitro and in vivo, in part by reducing NF-κB transcriptional activity. We investigated the hypothesis that the protective effect of KDACi involves transcriptional regulation of microRNAs (miRs), potential new targets in diabetes treatment. Insulin-producing INS1 cells were cultured with or without the broad-spectrum KDACi Givinostat, prior to exposure to the pro-inflammatory cytokines IL-1ß and IFN-γ for 6 h or 24 h, and miR expression was profiled with miR array. Thirteen miRs (miR-7a-2-3p, miR-29c-3p, miR-96-5p, miR-101a-3p, miR-140-5p, miR-146a-5p, miR-146b-5p, miR-340-5p, miR-384-5p, miR-455-5p, miR-466b-2-3p, miR-652-5p, and miR-3584-5p) were regulated by both cytokines and Givinostat, and nine were examined by qRT-PCR. miR-146a-5p was strongly regulated by cytokines and KDACi and was analyzed further. miR-146a-5p expression was induced by cytokines in rat and human islets. Cytokine-induced miR-146a-5p expression was specific for INS1 and ß-TC3 cells, whereas α-TC1 cells exhibited a higher basal expression. Transfection of INS1 cells with miR-146a-5p reduced cytokine signaling, including the activity of NF-κB and iNOS promoters, as well as NO production and protein levels of iNOS and its own direct targets TNF receptor associated factor 6 (TRAF6) and interleukin-1 receptor-associated kinase 1 (IRAK1). miR-146a-5p was elevated in the pancreas of diabetes-prone BB-DP rats at diabetes onset, suggesting that miR-146a-5p could play a role in type 1 diabetes development. The miR array of cytokine-exposed INS1 cells rescued by KDACi revealed several other miRs potentially involved in cytokine-induced ß-cell apoptosis, demonstrating the strength of this approach.

The EndoC-ßH1 cell line is a valid model of human beta cells and applicable for screenings to identify novel drug target candidates.

OBJECTIVE: To characterize the EndoC-ßH1 cell line as a model for human beta cells and evaluate its beta cell functionality, focusing on insulin secretion, proliferation, apoptosis and ER stress, with the objective to assess its potential as a screening platform for identification of novel anti-diabetic drug candidates. METHODS: EndoC-ßH1 was transplanted into mice for validation of in vivo functionality. Insulin secretion was evaluated in cells cultured as monolayer and as pseudoislets, as well as in diabetic mice. Cytokine induced apoptosis, glucolipotoxicity, and ER stress responses were assessed. Beta cell relevant mRNA and protein expression were investigated by qPCR and antibody staining. Hundreds of proteins or peptides were tested for their effect on insulin secretion and proliferation. RESULTS: Transplantation of EndoC-ßH1 cells restored normoglycemia in streptozotocin induced diabetic mice. Both in vitro and in vivo, we observed a clear insulin response to glucose, and, in vitro, we found a significant increase in insulin secretion from EndoC-ßH1 pseudoislets compared to monolayer cultures for both glucose and incretins. Apoptosis and ER stress were inducible in the cells and caspase 3/7 activity was elevated in response to cytokines, but not affected by the saturated fatty acid palmitate. By screening of various proteins and peptides, we found Bombesin (BB) receptor agonists and Pituitary Adenylate Cyclase-Activating Polypeptides (PACAP) to significantly induce insulin secretion and the proteins SerpinA6, STC1, and APOH to significantly stimulate proliferation. ER stress was readily induced by Tunicamycin and resulted in a reduction of insulin mRNA. Somatostatin (SST) was found to be expressed by 1% of the cells and manipulation of the SST receptors was found to significantly affect insulin secretion. CONCLUSIONS: Overall, the EndoC-ßH1 cells strongly resemble human islet beta cells in terms of glucose and incretin stimulated insulin secretion capabilities. The cell line has an active cytokine induced caspase 3/7 apoptotic pathway and is responsive to ER stress initiation factors. The cells' ability to proliferate can be further increased by already known compounds as well as by novel peptides and proteins. Based on its robust performance during the functionality assessment assays, the EndoC-ßH1 cell line was successfully used as a screening platform for identification of novel anti-diabetic drug candidates.

Analysis artefacts of the INS-IGF2 fusion transcript.

BACKGROUND: In gene expression analysis, overlapping genes, splice variants, and fusion transcripts are potential sources of data analysis artefacts, depending on how the observed intensity is assigned to one, or more genes. We here exemplify this by an in-depth analysis of the INS-IGF2 fusion transcript, which has recently been reported to be among the highest expressed transcripts in human pancreatic beta cells and its protein indicated as a novel autoantigen in Type 1 Diabetes. RESULTS: Through RNA sequencing and variant specific qPCR analyses we demonstrate that the true abundance of INS-IGF2 is >20,000 fold lower than INS in human beta cells, and we suggest an explanation to the nature of the artefacts which have previously led to overestimation of the gene expression level in selected studies. We reinvestigated the previous reported findings of detection of INS-IGF2 using antibodies both in Western blotting and immunohistochemistry. We found that the one available commercial antibody (BO1P) raised against recombinant INS-IGF2 show strong cross-reaction to native proinsulin, and we did not detect INS-IGF2 protein in the human beta cell line EndoC-ßH1. Furthermore, using highly sensitive proteomics analysis we could not demonstrate INS-IGF2 protein in samples of human islets nor in EndoC-ßH1. CONCLUSIONS: Sequence features, such as fusion transcripts spanning multiple genes can lead to unexpected results in gene expression analysis, and care must be taken in generating and interpreting the results. For the specific case of INS-IGF2 we conclude that the abundance of the fusion transcript/protein is exceedingly lower than previously reported, and that current immuno-reagents available for detecting INS-IGF2 protein have a strong cross-reaction to native human proinsulin. Finally, we were unable to detect INS-IGF2 protein by proteomics analysis.

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

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.

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

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.

Partial promoter substitutions generating transcriptional sentinels of diverse signaling pathways in embryonic stem cells and mice.

Extracellular signals in development, physiology, homeostasis and disease often act by regulating transcription. Herein we describe a general method and specific resources for determining where and when such signaling occurs in live animals and for systematically comparing the timing and extent of different signals in different cellular contexts. We used recombinase-mediated cassette exchange (RMCE) to test the effect of successively deleting conserved genomic regions of the ubiquitously active Rosa26 promoter and substituting the deleted regions for regulatory sequences that respond to diverse extracellular signals. We thereby created an allelic series of embryonic stem cells and mice, each containing a signal-responsive sentinel with different fluorescent reporters that respond with sensitivity and specificity to retinoic acids, bone morphogenic proteins, activin A, Wnts or Notch, and that can be adapted to any pathway that acts via DNA elements.

Suppressor of cytokine signalling-3 inhibits Tumor necrosis factor-alpha induced apoptosis and signalling in beta cells.

Tumor necrosis factor-alpha (TNFalpha) is a pro-inflammatory cytokine involved in the pathogenesis of several diseases including type 1 diabetes mellitus (T1DM). TNFalpha in combination with interleukin-1-beta (IL-1beta) and/or interferon-gamma (IFNgamma) induces specific destruction of the pancreatic insulin-producing beta cells. Suppressor of cytokine signalling-3 (SOCS-3) proteins regulate signalling induced by a number of cytokines including growth hormone, IFNgamma and IL-1beta which signals via very distinctive pathways. The objective of this study was to investigate the effect of SOCS-3 on TNFalpha-induced signalling in beta cells. We found that apoptosis induced by TNFalpha alone or in combination with IL-1beta was suppressed by expression of SOCS-3 in the beta cell line INSr3#2. SOCS-3 inhibited TNFalpha-induced phosphorylation of the mitogen activated protein kinases ERK1/2, p38 and JNK in INSr3#2 cells and in primary rat islets. Furthermore, SOCS-3 repressed TNFalpha-induced degradation of IkappaB, NFkappaB DNA binding and transcription of the NFkappaB-dependent MnSOD promoter. Finally, expression of Socs-3 mRNA was induced by TNFalpha in rat islets in a transient manner with maximum expression after 1-2h. The ability of SOCS-3 to regulate signalling induced by the three major pro-inflammatory cytokines involved in the pathogenesis of T1DM makes SOCS-3 an interesting therapeutic candidate for protection of the beta cell mass.