Search

BACKGROUND: Identification of islet β cell death prior to the onset of type 1 diabetes (T1D) or type 2 diabetes (T2D) might allow for interventions to protect β cells and reduce diabetes risk. Circulating unmethylated DNA fragments arising from the human INS gene have been proposed as biomarkers of β cell death, but this gene alone may not be sufficiently specific to report β cell death. RESULTS: To identify new candidate genes whose CpG sites may show greater specificity for β cells, we performed unbiased DNA methylation analysis using the Infinium HumanMethylation 450 array on 64 human islet preparations and 27 non-islet human tissues. For verification of array results, bisulfite DNA sequencing of human β cells and 11 non-β cell tissues was performed on 5 of the top 10 CpG sites that were found to be differentially methylated. We identified the CHTOP gene as a candidate whose CpGs show a greater frequency of unmethylation in human islets. A digital PCR strategy was used to determine the methylation pattern of CHTOP and INS CpG sites in primary human tissues. Although both INS and CHTOP contained unmethylated CpG sites in non-islet tissues, they occurred in a non-overlapping pattern. Based on Naïve Bayes classifier analysis, the two genes together report 100% specificity for islet damage. Digital PCR was then performed on cell-free DNA from serum from human subjects. Compared to healthy controls (N = 10), differentially methylated CHTOP and INS levels were higher in youth with new onset T1D (N = 43) and, unexpectedly, in healthy autoantibody-negative youth who have first-degree relatives with T1D (N = 23). When tested in lean (N = 32) and obese (N = 118) youth, increased levels of unmethylated INS and CHTOP were observed in obese individuals. CONCLUSION: Our data suggest that concurrent measurement of circulating unmethylated INS and CHTOP has the potential to detect islet death in youth at risk for both T1D and T2D. Our data also support the use of multiple parame, SCOPUS: ar.j, info:eu-repo/semantics/published

info:eu-repo/semantics/published

The autoimmune-mediated beta-cell death in type 1 diabetes (T1DM) is associated with local inflammation (insulitis). We examined the role of MCPIP1 (monocyte chemotactic protein–induced protein 1), a novel cytokine-induced antiinflammatory protein, in this process. Basal MCPIP1 expression was lower in rat vs. human islets and beta-cells. Proinflammatory cytokines stimulated MCPIP1 expression in rat and human islets and in insulin-secreting cells. Moderate overexpression of MCPIP1 protected insulin-secreting INS1E cells against cytokine toxicity by a mechanism dependent on the presence of the PIN/DUB domain in MCPIP1. It also reduced cytokine-induced Chop and C/ebpβ expression and maintained MCL-1 expression. The shRNA-mediated suppression of MCPIP1 led to the potentiation of cytokine-mediated NFκB activation and cytokine toxicity in human EndoC-βH1 beta-cells. MCPIP1 expression was very high in infiltrated beta-cells before and after diabetes manifestation in the LEW.1AR1-iddm rat model of human T1DM. The extremely high expression of MCPIP1 in clonal beta-cells was associated with a failure of the regulatory feedback-loop mechanism, ER stress induction and high cytokine toxicity. In conclusion, our data indicate that the expression level of MCPIP1 affects the susceptibility of insulin-secreting cells to cytokines and regulates the mechanism of beta-cell death in T1DM., SCOPUS: ar.j, info:eu-repo/semantics/published

info:eu-repo/semantics/published

Pancreatic β-cell dysfunction and death are determinant events in type 1 diabetes (T1D), but the molecular mechanisms behind β-cell fate remain poorly understood. Alternative splicing is a post-translational mechanism by which a single gene generates different mRNA and protein isoforms, expanding the transcriptome complexity and enhancing protein diversity. Neuron-specific and certain serine/arginine-rich RNA binding proteins (RBP) are enriched in β-cells, playing crucial roles in the regulation of insulin secretion and β-cell survival. Moreover, alternative exon networks, regulated by inflammation or diabetes susceptibility genes, control key pathways and processes for the correct function and survival of β-cells. The challenge ahead of us is to understand the precise role of alternative splicing regulators and splice variants on β-cell function, dysfunction and death and develop tools to modulate it., “Innovative Medicines Initiative 2”;“European Union(EU)”“Horizon2020”; and EFPIA”,“JDRF International” and The Leona M.and Harry B.Helmsley Charitable Trust”, SCOPUS: re.j, info:eu-repo/semantics/published

info:eu-repo/semantics/published

Our current understanding of the pathogenesis of type 1 diabetes (T1D) arose, in large part, from studies using the non-obese diabetic (NOD) mouse model. In the present study, we chose a human-focused method to investigate T1D disease mechanisms and potential targets for therapeutic intervention by directly analysing human donor pancreatic islets from individuals with T1D., SCOPUS: ar.j, info:eu-repo/semantics/published

Pancreatic beta cell failure is the central event leading to diabetes. Beta cells share many phenotypic traits with neurons, and proper beta cell function relies on the activation of several neuron-like transcription programs. Regulation of gene expression by alternative splicing plays a pivotal role in brain, where it affects neuronal development, function, and disease. The role of alternative splicing in beta cells remains unclear, but recent data indicate that splicing alterations modulated by both inflammation and susceptibility genes for diabetes contribute to beta cell dysfunction and death. Here we used RNA sequencing to compare the expression of splicing-regulatory RNA-binding proteins in human islets, brain, and other human tissues, and we identified a cluster of splicing regulators that are expressed in both beta cells and brain. Four of them, namely Elavl4, Nova2, Rbox1, and Rbfox2, were selected for subsequent functional studies in insulin-producing rat INS-1E, human EndoC-βH1 cells, and in primary rat beta cells. Silencing of Elavl4 and Nova2 increased beta cell apoptosis, whereas silencing of Rbfox1 and Rbfox2 increased insulin content and secretion. Interestingly, Rbfox1 silencing modulates the splicing of the actin-remodeling protein gelsolin, increasing gelsolin expression and leading to faster glucose-induced actin depolymerization and increased insulin release. Taken together, these findings indicate that beta cells share common splicing regulators and programs with neurons. These splicing regulators play key roles in insulin release and beta cell survival, and their dysfunction may contribute to the loss of functional beta cell mass in diabetes., SCOPUS: ar.j, info:eu-repo/semantics/published

There are presently no reliable ways to quantify endocrine cell mass (ECM) in vivo, which prevents an accurate understanding of the progressive beta cell loss in diabetes or following islet transplantation. To address this unmet need, we coupled RNA sequencing of human pancreatic islets to a systems biology approach to identify new biomarkers of the endocrine pancreas. Dipeptidyl-Peptidase 6 (DPP6) was identified as a target whose mRNA expression is at least 25-fold higher in human pancreatic islets as compared to surrounding tissues and is not changed by proinflammatory cytokines. At the protein level, DPP6 localizes only in beta and alpha cells within the pancreas. We next generated a high-affinity camelid single-domain antibody (nanobody) targeting human DPP6. The nanobody was radiolabelled and in vivo SPECT/CT imaging and biodistribution studies were performed in immunodeficient mice that were either transplanted with DPP6-expressing Kelly neuroblastoma cells or insulin-producing human EndoC-βH1 cells. The human DPP6-expressing cells were clearly visualized in both models. In conclusion, we have identified a novel beta and alpha cell biomarker and developed a tracer for in vivo imaging of human insulin secreting cells. This provides a useful tool to non-invasively follow up intramuscularly implanted insulin secreting cells., SCOPUS: ar.j, info:eu-repo/semantics/published

The BCL-2 family are crucial regulators of the mitochondrial pathway of apoptosis in normal physiology and disease. Besides their role in cell death, BCL-2 proteins have been implicated in the regulation of mitochondrial oxidative phosphorylation and cellular metabolism. It remains unclear, however, whether these proteins have a physiological role in glucose homeostasis and metabolism in vivo Here we report that fat accumulation in the liver increases JNK-dependent BIM expression in hepatocytes. To determine the consequences of hepatic BIM deficiency in diet-induced obesity, we generated liver-specific BIM knockout (BLKO) mice. BLKO mice had lower hepatic lipid content, increased insulin signalling and improved global glucose metabolism. Consistent with these findings, lipogenic and lipid uptake genes were downregulated and lipid oxidation enhanced in obese BLKO mice. Mechanistically, BIM deficiency improved mitochondrial function and decreased oxidative stress, oxidation of protein tyrosine phosphatases and ameliorated activation of PPAR-γ/SREBP1/CD36 in hepatocytes from high fat fed mice. Importantly, short-term knockdown of BIM rescued obese mice from insulin resistance, evidenced by reduced fat accumulation and improved insulin sensitivity. Our data indicate that BIM is an important regulator of liver dysfunction in obesity, and a novel therapeutic target for restoring hepatocyte function., SCOPUS: ar.j, info:eu-repo/semantics/published

Progressive failure of insulin-producing beta cells is the central event leading to diabetes, but the signalling networks controlling beta cell fate remain poorly understood. Here we show that SRp55, a splicing factor regulated by the diabetes susceptibility gene GLIS3, has a major role in maintaining function and survival of human beta cells. RNA-seq analysis revealed that SRp55 regulates the splicing of genes involved in cell survival and death, insulin secretion and JNK signalling. Specifically, SRp55-mediated splicing changes modulate the function of the pro-apoptotic proteins BIM and BAX, JNK signalling and endoplasmic reticulum stress, explaining why SRp55 depletion triggers beta cell apoptosis. Furthermore, SRp55 depletion inhibits beta cell mitochondrial function, explaining the observed decrease in insulin release. These data unveil a novel layer of regulation of human beta cell function and survival, namely alternative splicing modulated by key splicing regulators such as SRp55 that may crosstalk with candidate genes for diabetes., SCOPUS: ar.j, info:eu-repo/semantics/published

info:eu-repo/semantics/published

info:eu-repo/semantics/nonPublished

The thermogenic activity of brown adipose tissue (BAT) and browning of white adipose tissue are important components of energy expenditure. Here we show that GPR120, a receptor for polyunsaturated fatty acids, promotes brown fat activation. Using RNA-seq to analyse mouse BAT transcriptome, we find that the gene encoding GPR120 is induced by thermogenic activation. We further show that GPR120 activation induces BAT activity and promotes the browning of white fat in mice, whereas GRP120-null mice show impaired cold-induced browning. Omega-3 polyunsaturated fatty acids induce brown and beige adipocyte differentiation and thermogenic activation, and these effects require GPR120. GPR120 activation induces the release of fibroblast growth factor-21 (FGF21) by brown and beige adipocytes, and increases blood FGF21 levels. The effects of GPR120 activation on BAT activation and browning are impaired in FGF21-null mice and cells. Thus, the lipid sensor GPR120 activates brown fat via a mechanism that involves induction of FGF21., SCOPUS: ar.j, info:eu-repo/semantics/published

info:eu-repo/semantics/nonPublished

JOURNAL ARTICLE, SCOPUS: ar.j, info:eu-repo/semantics/published

Alternative splicing (AS) is a fundamental mechanism for the regulation of gene expression. It affects more than 90% of human genes but its role in the regulation of pancreatic beta cells, the producers of insulin, remains unknown. Our recently published data indicated that the 'neuron-specific' Nova1 splicing factor is expressed in pancreatic beta cells. We have presently coupled specific knockdown (KD) of Nova1 with RNA-sequencing to determine all splice variants and downstream pathways regulated by this protein in beta cells. Nova1 KD altered the splicing of nearly 5000 transcripts. Pathway analysis indicated that these genes are involved in exocytosis, apoptosis, insulin receptor signaling, splicing and transcription. In line with these findings, Nova1 silencing inhibited insulin secretion and induced apoptosis basally and after cytokine treatment in rodent and human beta cells. These observations identify a novel layer of regulation of beta cell function, namely AS controlled by key splicing regulators such as Nova1., SCOPUS: ar.j, info:eu-repo/semantics/published

info:eu-repo/semantics/nonPublished

info:eu-repo/semantics/nonPublished

info:eu-repo/semantics/nonPublished

L'objectif de ce travail est de développer et d'évaluer des approches méthodologiques pour laprédiction de séquences cis-régulatrices. Ces approches ont été intégrées dans la suite logicielleRSAT (Regulatory Sequences Analysis Tools). Ces séquences jouent un rôle important dans larégulation de l'expression des gènes. Cette régulation, au niveau transcriptionnel, s'effectue àtravers la reconnaissance spécifique entre les facteurs de transcription et leurs sites de fixation(TFBS) au niveau de l'ADN.Nous avons développé et évalué une série d'outils bioinformatiques qui utilisent les matricesposition-poids pour prédire les TFBS ainsi que les modules cis-régulateurs (CRM). Nos outilsprésentent l'avantage d'intégrer les différentes approches déjà proposées par d'autres auteurs touten proposant des fonctionnalités innovantes.Nous proposons notamment une nouvelle approche pour la prédiction de CRM basé sur ladétection de régions significativement enrichies en TFBS. Nous les avons appelés les CRER (pourCis-Regulatory Elements Enriched Regions). Un autre aspect essentiel de toute notre approcheréside dans le fait que nous proposons des mesures statistiques rigoureuses pour estimerthéoriquement et empiriquement le risque associé aux différentes prédictions. Les méthodes deprédictions de séquences cis-regulatrices prédisent en effet un taux de fausses prédictionsgénéralement élevé. Nous intégrons un calcul des P-valeurs associées à toutes les prédictions.Nous proposons ainsi une mesure fiable de la probabilité de faux positifs.Nous avons appliqué nos outils pour une évaluation systématique de l'effet du modèle debackground sur la précision des prédictions à partir de la base de données de TRANSFAC. Nosrésultats suggèrent une grande variabilité pour les modèles qui optimisent la précision desprédictions. Il faut choisir le modèle de background au cas par cas selon la matrice considérée.Nous avons ensuite évalué la, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

This protocol shows how to detect putative cis-regulatory elements and regions enriched in such elements with the regulatory sequence analysis tools (RSAT) web server (http://rsat.ulb.ac.be/rsat/). The approach applies to known transcription factors, whose binding specificity is represented by position-specific scoring matrices, using the program matrix-scan. The detection of individual binding sites is known to return many false predictions. However, results can be strongly improved by estimating P value, and by searching for combinations of sites (homotypic and heterotypic models). We illustrate the detection of sites and enriched regions with a study case, the upstream sequence of the Drosophila melanogaster gene even-skipped. This protocol is also tested on random control sequences to evaluate the reliability of the predictions. Each task requires a few minutes of computation time on the server. The complete protocol can be executed in about one hour., Comparative Study, Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

Transcriptional regulation plays an essential role in all steps of morphogenesis, by controlling the specific subsets of genes that will be expressed in different cell types, and at different times during embryonic development. The control of gene expression is also crucial to maintain the basic cellular functions (e.g. cell divisions) and the response of the organism to its environment (e.g. metabolic regulation). The spatio-temporal control of gene expression is ensured by interactions between transcription factors and specific loci, called cis-acting regulatory elements., SCOPUS: ch.b, info:eu-repo/semantics/published