Mariana Igoillo-Esteve

DNA methylation profiling identifies epigenetic dysregulation in pancreatic islets from type 2 diabetic patients

In addition to genetic predisposition, environmental and lifestyle factors contribute to the pathogenesis of type 2 diabetes (T2D). Epigenetic changes may provide the link for translating environmental exposures into pathological mechanisms. In this study, we performed the first comprehensive DNA methylation profiling in pancreatic islets from T2D and non‐diabetic donors. We uncovered 276 CpG loci affiliated to promoters of 254 genes displaying significant differential DNA methylation in diabetic islets. These methylation changes were not present in blood cells from T2D individuals nor were they experimentally induced in non‐diabetic islets by exposure to high glucose. For a subgroup of the differentially methylated genes, concordant transcriptional changes were present. Functional annotation of the aberrantly methylated genes and RNAi experiments highlighted pathways implicated in β‐cell survival and function; some are implicated in cellular dysfunction while others facilitate adaptation to stressors. Together, our findings offer new insights into the intricate mechanisms of T2D pathogenesis, underscore the important involvement of epigenetic dysregulation in diabetic islets and may advance our understanding of T2D aetiology.

The Human Pancreatic Islet Transcriptome: Expression of Candidate Genes for Type 1 Diabetes and the Impact of Pro-Inflammatory Cytokines

Type 1 diabetes (T1D) is an autoimmune disease in which pancreatic beta cells are killed by infiltrating immune cells and by cytokines released by these cells. Signaling events occurring in the pancreatic beta cells are decisive for their survival or death in diabetes. We have used RNA sequencing (RNA–seq) to identify transcripts, including splice variants, expressed in human islets of Langerhans under control conditions or following exposure to the pro-inflammatory cytokines interleukin-1β (IL-1β) and interferon-γ (IFN-γ). Based on this unique dataset, we examined whether putative candidate genes for T1D, previously identified by GWAS, are expressed in human islets. A total of 29,776 transcripts were identified as expressed in human islets. Expression of around 20% of these transcripts was modified by pro-inflammatory cytokines, including apoptosis- and inflammation-related genes. Chemokines were among the transcripts most modified by cytokines, a finding confirmed at the protein level by ELISA. Interestingly, 35% of the genes expressed in human islets undergo alternative splicing as annotated in RefSeq, and cytokines caused substantial changes in spliced transcripts. Nova1, previously considered a brain-specific regulator of mRNA splicing, is expressed in islets and its knockdown modified splicing. 25/41 of the candidate genes for T1D are expressed in islets, and cytokines modified expression of several of these transcripts. The present study doubles the number of known genes expressed in human islets and shows that cytokines modify alternative splicing in human islet cells. Importantly, it indicates that more than half of the known T1D candidate genes are expressed in human islets. This, and the production of a large number of chemokines and cytokines by cytokine-exposed islets, reinforces the concept of a dialog between pancreatic islets and the immune system in T1D. This dialog is modulated by candidate genes for the disease at both the immune system and beta cell level.

Glucagon-Like Peptide-1 Agonists Protect Pancreatic β-Cells From Lipotoxic Endoplasmic Reticulum Stress Through Upregulation of BiP and JunB

OBJECTIVE Chronic exposure of pancreatic β-cells to saturated free fatty acids (FFAs) causes endoplasmic reticulum (ER) stress and apoptosis and may contribute to β-cell loss in type 2 diabetes. Here, we evaluated the molecular mechanisms involved in the protection of β-cells from lipotoxic ER stress by glucagon-like peptide (GLP)-1 agonists utilized in the treatment of type 2 diabetes. RESEARCH DESIGN AND METHODS INS-1E or fluorescence-activated cell sorter–purified primary rat β-cells were exposed to oleate or palmitate with or without the GLP-1 agonist exendin-4 or forskolin. Cyclopiazonic acid was used as a synthetic ER stressor, while the activating transcription factor 4–C/EBP homologous protein branch was selectively activated with salubrinal. The ER stress signaling pathways modulated by GLP-1 agonists were studied by real-time PCR and Western blot. Knockdown by RNA interference was used to identify mediators of the antiapoptotic GLP-1 effects in the ER stress response and downstream mitochondrial cell death mechanisms. RESULTS Exendin-4 and forskolin protected β-cells against FFAs via the induction of the ER chaperone BiP and the antiapoptotic protein JunB that mediate β-cell survival under lipotoxic conditions. On the other hand, exendin-4 and forskolin protected against synthetic ER stressors by inactivating caspase 12 and upregulating Bcl-2 and X-chromosome–linked inhibitor of apoptosis protein that inhibit mitochondrial apoptosis. CONCLUSIONS These observations suggest that GLP-1 agonists increase in a context-dependent way the β-cell defense mechanisms against different pathways involved in ER stress–induced apoptosis. The identification of the pathways modulated by GLP-1 agonists allows for targeted approaches to alleviate β-cell ER stress in diabetes.

RNA-sequencing identifies dysregulation of the human pancreatic islet transcriptome by the saturated fatty acid palmitate

Pancreatic β-cell dysfunction and death are central in the pathogenesis of type 2 diabetes (T2D). Saturated fatty acids cause β-cell failure and contribute to diabetes development in genetically predisposed individuals. Here we used RNA sequencing to map transcripts expressed in five palmitate-treated human islet preparations, observing 1,325 modified genes. Palmitate induced fatty acid metabolism and endoplasmic reticulum (ER) stress. Functional studies identified novel mediators of adaptive ER stress signaling. Palmitate modified genes regulating ubiquitin and proteasome function, autophagy, and apoptosis. Inhibition of autophagic flux and lysosome function contributed to lipotoxicity. Palmitate inhibited transcription factors controlling β-cell phenotype, including PAX4 and GATA6. Fifty-nine T2D candidate genes were expressed in human islets, and 11 were modified by palmitate. Palmitate modified expression of 17 splicing factors and shifted alternative splicing of 3,525 transcripts. Ingenuity Pathway Analysis of modified transcripts and genes confirmed that top changed functions related to cell death. Database for Annotation, Visualization and Integrated Discovery (DAVID) analysis of transcription factor binding sites in palmitate-modified transcripts revealed a role for PAX4, GATA, and the ER stress response regulators XBP1 and ATF6. This human islet transcriptome study identified novel mechanisms of palmitate-induced β-cell dysfunction and death. The data point to cross talk between metabolic stress and candidate genes at the β-cell level.

STAT1 is a master regulator of pancreatic beta-cell apoptosis and islet inflammation.

Cytokines produced by islet-infiltrating immune cells induce β-cell apoptosis in type 1 diabetes. The IFN-γ-regulated transcription factors STAT1/IRF-1 have apparently divergent effects on β-cells. Thus, STAT1 promotes apoptosis and inflammation, whereas IRF-1 down-regulates inflammatory mediators. To understand the molecular basis for these differential outcomes within a single signal transduction pathway, we presently characterized the gene networks regulated by STAT1 and IRF-1 in β-cells. This was done by using siRNA approaches coupled to microarray analysis of insulin-producing cells exposed or not to IL-1β and IFN-γ. Relevant microarray findings were further studied in INS-1E cells and primary rat β-cells. STAT1, but not IRF-1, mediates the cytokine-induced loss of the differentiated β-cell phenotype, as indicated by decreased insulin, Pdx1, MafA, and Glut2. Furthermore, STAT1 regulates cytokine-induced apoptosis via up-regulation of the proapoptotic protein DP5. STAT1 and IRF-1 have opposite effects on cytokine-induced chemokine production, with IRF-1 exerting negative feedback inhibition on STAT1 and downstream chemokine expression. The present study elucidates the transcriptional networks through which the IFN-γ/STAT1/IRF-1 axis controls β-cell function/differentiation, demise, and islet inflammation.

Causes and cures for endoplasmic reticulum stress in lipotoxic β-cell dysfunction.

Pancreatic β‐cell dysfunction is central to the pathogenesis of type 2 diabetes, and the loss of functional β‐cell mass in type 2 diabetes is at least in part secondary to increased β‐cell apoptosis. Accumulating evidence suggests that endoplasmic reticulum (ER) stress is present in β‐cells in type 2 diabetes. Free fatty acids (FFAs) cause ER stress and are putative mediators of β‐cell dysfunction and death. In this review, we discuss the molecular mechanisms underlying ER stress induced by saturated and unsaturated FFAs. Oleate and palmitate trigger ER stress through ER Ca2+ depletion and build‐up of unfolded proteins in the secretory pathway. Saturated and unsaturated FFAs elicit a differential signal transduction in the three branches of the ER stress response, resulting in different survival/apoptosis outcomes. The protection of β‐cells against FFAs through the interference with ER stress signalling has opened novel therapeutic perspectives for type 2 diabetes. Chemical chaperones, salubrinal and glucagon‐like peptide‐1 (GLP‐1) analogues have been used to protect β‐cells from lipotoxic ER stress. Importantly, the pro‐ and antiapoptotic effects of these compounds are cell and context dependent.

Death Protein 5 and p53-Upregulated Modulator of Apoptosis Mediate the Endoplasmic Reticulum Stress–Mitochondrial Dialog Triggering Lipotoxic Rodent and Human β-Cell Apoptosis

Environmental factors such as diets rich in saturated fats contribute to dysfunction and death of pancreatic β-cells in diabetes. Endoplasmic reticulum (ER) stress is elicited in β-cells by saturated fatty acids. Here we show that palmitate-induced β-cell apoptosis is mediated by the intrinsic mitochondrial pathway. By microarray analysis, we identified a palmitate-triggered ER stress gene expression signature and the induction of the BH3-only proteins death protein 5 (DP5) and p53-upregulated modulator of apoptosis (PUMA). Knockdown of either protein reduced cytochrome c release, caspase-3 activation, and apoptosis in rat and human β-cells. DP5 induction depends on inositol-requiring enzyme 1 (IRE1)–dependent c-Jun NH2-terminal kinase and PKR–like ER kinase (PERK)–induced activating transcription factor (ATF3) binding to its promoter. PUMA expression is also PERK/ATF3-dependent, through tribbles 3 (TRB3)–regulated AKT inhibition and FoxO3a activation. DP5−/− mice are protected from high fat diet–induced loss of glucose tolerance and have twofold greater pancreatic β-cell mass. This study elucidates the crosstalk between lipotoxic ER stress and the mitochondrial pathway of apoptosis that causes β-cell death in diabetes.

An update on lipotoxic endoplasmic reticulum stress in pancreatic β-cells

The UPR (unfolded protein response) or ER (endoplasmic reticulum) stress response was first described 20 years ago. The field of ER stress has expanded tremendously since, moving from basic biology in yeast to human neurodegenerative, inflammatory, cardiovascular and neoplastic diseases. The ER stress response has also been implicated in diabetes development, affecting both insulin production by pancreatic beta-cells and insulin sensitivity in peripheral tissues. In the present mini-review, we focus on recent progress in the field of ER stress in pancreatic beta-cells. Recent advances in the understanding of lipotoxic ER stress and beta-cell recovery from ER stress are discussed.

Ubiquitin Fold Modifier 1 (UFM1) and Its Target UFBP1 Protect Pancreatic Beta Cells from ER Stress-Induced Apoptosis

UFM1 is a member of the ubiquitin like protein family. While the enzymatic cascade of UFM1 conjugation has been elucidated in recent years, the biological function remains largely unknown. In this report we demonstrate that the recently identified C20orf116 [1], which we name UFM1-binding protein 1 containing a PCI domain (UFBP1), andCDK5RAP3 interact with UFM1. Components of the UFM1 conjugation pathway (UFM1, UFBP1, UFL1 and CDK5RAP3) are highly expressed in pancreatic islets of Langerhans and some other secretory tissues. Co-localization of UFM1 with UFBP1 in the endoplasmic reticulum (ER)depends on UFBP1. We demonstrate that ER stress, which is common in secretory cells, induces expression of Ufm1, Ufbp1 and Ufl1 in the beta-cell line INS-1E.siRNA-mediated Ufm1 or Ufbp1knockdown enhances apoptosis upon ER stress.Silencing the E3 enzyme UFL1, results in similar outcomes, suggesting that UFM1-UFBP1 conjugation is required to prevent ER stress-induced apoptosis. Together, our data suggest that UFM1-UFBP1participate in preventing ER stress-induced apoptosis in protein secretory cells.

Cytokines induce endoplasmic reticulum stress in human, rat and mouse beta cells via different mechanisms

Aims/hypothesisProinflammatory cytokines contribute to beta cell damage in type 1 diabetes in part through activation of endoplasmic reticulum (ER) stress. In rat beta cells, cytokine-induced ER stress involves NO production and consequent inhibition of the ER Ca2+ transporting ATPase sarco/endoplasmic reticulum Ca2+ pump 2 (SERCA2B). However, the mechanisms by which cytokines induce ER stress and apoptosis in mouse and human pancreatic beta cells remain unclear. The purpose of this study is to elucidate the role of ER stress on cytokine-induced beta cell apoptosis in these three species and thus solve ongoing controversies in the field.MethodsRat and mouse insulin-producing cells, human pancreatic islets and human EndoC-βH1 cells were exposed to the cytokines IL-1β, TNF-α and IFN-γ, with or without NO inhibition. A global comparison of cytokine-modulated gene expression in human, mouse and rat beta cells was also performed. The chemical chaperone tauroursodeoxycholic acid (TUDCA) and suppression of C/EBP homologous protein (CHOP) were used to assess the role of ER stress in cytokine-induced apoptosis of human beta cells.ResultsNO plays a key role in cytokine-induced ER stress in rat islets, but not in mouse or human islets. Bioinformatics analysis indicated greater similarity between human and mouse than between human and rat global gene expression after cytokine exposure. The chemical chaperone TUDCA and suppression of CHOP or c-Jun N-terminal kinase (JNK) protected human beta cells against cytokine-induced apoptosis.Conclusions/interpretationThese observations clarify previous results that were discrepant owing to the use of islets from different species, and confirm that cytokine-induced ER stress contributes to human beta cell death, at least in part via JNK activation.