Sonia Gattesco

Alterations in MicroRNA Expression Contribute to Fatty Acid–Induced Pancreatic β-Cell Dysfunction

OBJECTIVE—Visceral obesity and elevated plasma free fatty acids are predisposing factors for type 2 diabetes. Chronic exposure to these lipids is detrimental for pancreatic β-cells, resulting in reduced insulin content, defective insulin secretion, and apoptosis. We investigated the involvement in this phenomenon of microRNAs (miRNAs), a class of noncoding RNAs regulating gene expression by sequence-specific inhibition of mRNA translation. RESEARCH DESIGN AND METHODS—We analyzed miRNA expression in insulin-secreting cell lines or pancreatic islets exposed to palmitate for 3 days and in islets from diabetic db/db mice. We studied the signaling pathways triggering the changes in miRNA expression and determined the impact of the miRNAs affected by palmitate on insulin secretion and apoptosis. RESULTS—Prolonged exposure of the β-cell line MIN6B1 and pancreatic islets to palmitate causes a time- and dose-dependent increase of miR34a and miR146. Elevated levels of these miRNAs are also observed in islets of diabetic db/db mice. miR34a rise is linked to activation of p53 and results in sensitization to apoptosis and impaired nutrient-induced secretion. The latter effect is associated with inhibition of the expression of vesicle-associated membrane protein 2, a key player in β-cell exocytosis. Higher miR146 levels do not affect the capacity to release insulin but contribute to increased apoptosis. Treatment with oligonucleotides that block miR34a or miR146 activity partially protects palmitate-treated cells from apoptosis but is insufficient to restore normal secretion. CONCLUSIONS—Our findings suggest that at least part of the detrimental effects of palmitate on β-cells is caused by alterations in the level of specific miRNAs.

Involvement of MicroRNAs in the Cytotoxic Effects Exerted by Proinflammatory Cytokines on Pancreatic β-Cells

OBJECTIVE Pancreatic β-cells exposed to proinflammatory cytokines display alterations in gene expression resulting in defective insulin secretion and apoptosis. MicroRNAs are small noncoding RNAs emerging as key regulators of gene expression. Here, we evaluated the contribution of microRNAs to cytokine-mediated β-cell cytotoxicity. RESEARCH DESIGN AND METHODS We used global microarray profiling and real-time PCR analysis to detect changes in microRNA expression in β-cells exposed to cytokines and in islets of pre-diabetic NOD mice. We assessed the involvement of the microRNAs affected in cytokine-mediated β-cell failure by modifying their expression in insulin-secreting MIN6 cells. RESULTS We found that IL-1β and TNF-α induce the expression of miR-21, miR-34a, and miR-146a both in MIN6 cells and human pancreatic islets. We further show an increase of these microRNAs in islets of NOD mice during development of pre-diabetic insulitis. Blocking miR-21, miR-34a, or miR-146a function using antisense molecules did not restore insulin-promoter activity but prevented the reduction in glucose-induced insulin secretion observed upon IL-1β exposure. Moreover, anti–miR-34a and anti–miR-146a treatment protected MIN6 cells from cytokine-triggered cell death. CONCLUSIONS Our data identify miR-21, miR-34a, and miR-146a as novel players in β-cell failure elicited in vitro and in vivo by proinflammatory cytokines, notably during the development of peri-insulitis that precedes overt diabetes in NOD mice.

Regulation of the expression of components of the exocytotic machinery of insulin-secreting cells by microRNAs.

Abstract Fine-tuning of insulin secretion from pancreatic β-cells participates in blood glucose homeostasis. Defects in this process can lead to chronic hyperglycemia and diabetes mellitus. Several proteins controlling insulin exocytosis have been identified, but the mechanisms regulating their expression remain poorly understood. Here, we show that two non-coding microRNAs, miR124a and miR96, modulate the expression of proteins involved in insulin exocytosis and affect secretion of the β-cell line MIN6B1. miR124a increases the levels of SNAP25, Rab3A and synapsin-1A and decreases those of Rab27A and Noc2. Inhibition of Rab27A expression is mediated by direct binding to the 3′-untranslated region of Rab27A mRNA. The effect on the other genes is indirect and linked to changes in mRNA levels. Over-expression of miR124a leads to exaggerated hormone release under basal conditions and a reduction in glucose-induced secretion. miR96 increases mRNA and protein levels of granuphilin, a negative modulator of insulin exocytosis, and decreases the expression of Noc2, resulting in lower capacity of MIN6B1 cells to respond to secretagogues. Our data identify miR124a and miR96 as novel regulators of the expression of proteins playing a critical role in insulin exocytosis and in the release of other hormones and neurotransmitters.

Changes in MicroRNA Expression Contribute to Pancreatic β-Cell Dysfunction in Prediabetic NOD Mice

During the initial phases of type 1 diabetes, pancreatic islets are invaded by immune cells, exposing β-cells to proinflammatory cytokines. This unfavorable environment results in gene expression modifications leading to loss of β-cell functions. To study the contribution of microRNAs (miRNAs) in this process, we used microarray analysis to search for changes in miRNA expression in prediabetic NOD mice islets. We found that the levels of miR-29a/b/c increased in islets of NOD mice during the phases preceding diabetes manifestation and in isolated mouse and human islets exposed to proinflammatory cytokines. Overexpression of miR-29a/b/c in MIN6 and dissociated islet cells led to impairment in glucose-induced insulin secretion. Defective insulin release was associated with diminished expression of the transcription factor Onecut2, and a consequent rise of granuphilin, an inhibitor of β-cell exocytosis. Overexpression of miR-29a/b/c also promoted apoptosis by decreasing the level of the antiapoptotic protein Mcl1. Indeed, a decoy molecule selectively masking the miR-29 binding site on Mcl1 mRNA protected insulin-secreting cells from apoptosis triggered by miR-29 or cytokines. Taken together, our findings suggest that changes in the level of miR-29 family members contribute to cytokine-mediated β-cell dysfunction occurring during the initial phases of type 1 diabetes.

Involvement of long non-coding RNAs in beta cell failure at the onset of type 1 diabetes in NOD mice

Aims/hypothesisExposure of pancreatic beta cells to cytokines released by islet-infiltrating immune cells induces alterations in gene expression, leading to impaired insulin secretion and apoptosis in the initial phases of type 1 diabetes. Long non-coding RNAs (lncRNAs) are a new class of transcripts participating in the development of many diseases. As little is known about their role in insulin-secreting cells, this study aimed to evaluate their contribution to beta cell dysfunction.MethodsThe expression of lncRNAs was determined by microarray in the MIN6 beta cell line exposed to proinflammatory cytokines. The changes induced by cytokines were further assessed by real-time PCR in islets of control and NOD mice. The involvement of selected lncRNAs modified by cytokines was assessed after their overexpression in MIN6 cells and primary islet cells.ResultsMIN6 cells were found to express a large number of lncRNAs, many of which were modified by cytokine treatment. The changes in the level of selected lncRNAs were confirmed in mouse islets and an increase in these lncRNAs was also seen in prediabetic NOD mice. Overexpression of these lncRNAs in MIN6 and mouse islet cells, either alone or in combination with cytokines, favoured beta cell apoptosis without affecting insulin production or secretion. Furthermore, overexpression of lncRNA-1 promoted nuclear translocation of nuclear factor of κ light polypeptide gene enhancer in B cells 1 (NF-κB).Conclusions/interpretationOur study shows that lncRNAs are modulated during the development of type 1 diabetes in NOD mice, and that their overexpression sensitises beta cells to apoptosis, probably contributing to their failure during the initial phases of the disease.

Murine Thy-1+ Dendritic Epidermal T Cell Lines Express Granule-Associated Perforin and a Family of Granzyme Molecules

Two T cell receptor gamma/delta + murine dendritic epidermal T cell (DETC) lines with cytotoxic potential towards various tumor cell lines are shown to express perforin and granzyme A both at the mRNA and protein levels. Furthermore, mRNA transcripts for granzyme B and at least one of the other granzymes D, E, F and G are detected in amounts equivalent to a murine IL-2-dependent alpha/beta + cytotoxic T lymphocyte cell line. Hemolytic granules containing serine-esterase (granzyme A) activity are isolated from a DETC line. Thus, cytolytically-active Thy-1+ DETC lines contain the granule-associated pore-forming protein, perforin, and at least one member of each of the three subgroups of granzyme serine esterases (granzyme A, B and D/E/F/G). These data support the proposed role of gamma/delta + DETC in immune surveillance, possibly exerting cytolytic functions against virus- or parasite-infected, transformed or stressed cells.

Involvement of the RNA-binding protein ARE/poly(U)-binding factor 1 (AUF1) in the cytotoxic effects of proinflammatory cytokines on pancreatic beta cells

Aims/hypothesisChronic exposure of pancreatic beta cells to proinflammatory cytokines leads to impaired insulin secretion and apoptosis. ARE/poly(U)-binding factor 1 (AUF1) belongs to a protein family that controls mRNA stability and translation by associating with adenosine- and uridine-rich regions of target messengers. We investigated the involvement of AUF1 in cytokine-induced beta cell dysfunction.MethodsProduction and subcellular distribution of AUF1 isoforms were analysed by western blotting. To test for their role in the control of beta cell functions, each isoform was overproduced individually in insulin-secreting cells. The contribution to cytokine-mediated beta cell dysfunction was evaluated by preventing the production of AUF1 isoforms by RNA interference. The effect of AUF1 on the production of potential targets was assessed by western blotting.ResultsMIN6 cells and human pancreatic islets were found to produce four AUF1 isoforms (p42>p45>p37>p40). AUF1 isoforms were mainly localised in the nucleus but were partially translocated to the cytoplasm upon exposure of beta cells to cytokines and activation of the ERK pathway. Overproduction of AUF1 did not affect glucose-induced insulin secretion but promoted apoptosis. This effect was associated with a decrease in the production of the anti-apoptotic proteins, B cell leukaemia/lymphoma 2 (BCL2) and myeloid cell leukaemia sequence 1 (MCL1). Silencing of AUF1 isoforms restored the levels of the anti-apoptotic proteins, attenuated the activation of the nuclear factor-κB (NFκB) pathway, and protected the beta cells from cytokine-induced apoptosis.Conclusions/interpretationOur findings point to a contribution of AUF1 to the deleterious effects of cytokines on beta cell functions and suggest a role for this RNA-binding protein in the early phases of type 1 diabetes.

Identification of islet-enriched long non-coding RNAs contributing to β-cell failure in type 2 diabetes

Objective Non-coding RNAs constitute a major fraction of the β-cell transcriptome. While the involvement of microRNAs is well established, the contribution of long non-coding RNAs (lncRNAs) in the regulation of β-cell functions and in diabetes development remains poorly understood. The aim of this study was to identify novel islet lncRNAs differently expressed in type 2 diabetes models and to investigate their role in β-cell failure and in the development of the disease. Methods Novel transcripts dysregulated in the islets of diet-induced obese mice were identified by high throughput RNA-sequencing coupled with de novo annotation. Changes in the level of the lncRNAs were assessed by real-time PCR. The functional role of the selected lncRNAs was determined by modifying their expression in MIN6 cells and primary islet cells. Results We identified about 1500 novel lncRNAs, a number of which were differentially expressed in obese mice. The expression of two lncRNAs highly enriched in β-cells, βlinc2, and βlinc3, correlated to body weight gain and glycemia levels in obese mice and was also modified in diabetic db/db mice. The expression of both lncRNAs was also modulated in vitro in isolated islet cells by glucolipotoxic conditions. Moreover, the expression of the human orthologue of βlinc3 was altered in the islets of type 2 diabetic patients and was associated to the BMI of the donors. Modulation of the level of βlinc2 and βlinc3 by overexpression or downregulation in MIN6 and mouse islet cells did not affect insulin secretion but increased β-cell apoptosis. Conclusions Taken together, the data show that lncRNAs are modulated in a model of obesity-associated type 2 diabetes and that variations in the expression of some of them may contribute to β-cell failure during the development of the disease.

O80 Le transfert des microRNAs représente un nouveau mode de communication entre les cellules bêta pancréatiques

Introduction Les microRNAs regulent l’expression genique a l’interieur des cellules les produisant, mais aussi dans des cellules receveuses suite a leur transfert par des microvesicules. Ce nouveau mode de communication intercellulaire est bien decrit dans certains systemes biologiques mais demeure peu caracterise dans les cellules beta pancreatiques. Dans la presente etude, nous avons analyse l’effet des conditions physiopathologiques associees au diabete sur le relâchement des microARNs par les cellules beta et determine l’impact biologique de leur transfert. Materiels et methodes Les microvesicules relâchees par des lignees insulinosecretrices et par des ilots pancreatiques ont ete isolees par ultra-centrifugation. Apres analyse de leur contenu, les microvesicules ont ete utilisees pour etudier l’impact du transfert des microARNs sur les fonctions des cellules beta receveuses. Resultats Des microARNs ont ete detectes dans les tous milieux de culture des cellules beta testees. L’analyse par micropuces a revele que le profil des microARNs relâches differe de celui des cellules. En effet, certains microARNs faiblement exprimes dans les cellules sont abondants dans les microvesicules et vice-versa. Ces resultats suggerent que certains microARNs sont preferentiellement relâches. De plus, l’incubation des cellules MIN6 en presence de cytokines ou de palmitate induit un changement dans les niveaux de plusieurs microARNs relâches. Fait interessant, l’incubation de MIN6 non-traitees en presence de microvesicules provenant du milieu de culture de MIN6 traitees aux cytokines conduit a une augmentation de l’apoptose et a une diminution de la proliferation des cellules receveuses. A l’oppose, les microvesicules purifiees du milieu de culture de MIN6 non-traitees n’affecte pas les fonctions des cellules receveuses. Conclusion L’ensemble des resultats obtenus suggerent que les cellules beta relâchent un groupe specifique de microARNs pouvant etre transferes a d’autres cellules beta et ainsi affecter leur fonction. Nos travaux supportent le concept que le transfert des microARNs constitue un mode de communication intercellulaire.

P168 - Rôle des microARNs dans le développement du diabète chez la souris NOD

Introduction Lors d’un diabete de type I, les cellules β pancreatiques sont les cibles d’une reaction auto-immunitaire. Suite a l’infiltration de macrophages et lymphocytes T dans les ilots pancreatiques, les cellules β sont exposees de facon prolongee a des cytokines pro-inflammatoires. Ces cytokines provoquent un dysfonctionnement des cellule β, en diminuant leur contenu et leur secretion d’insuline et en les sensibilisant a l’apoptose. Ici, nous avons etudie l’implication dans ces phenomenes physiopathologiques des microARNs, une famille de petits ARN non-codants qui regulent l’expression genique en inhibant la traduction de nombreux ARNs messagers. Materiels et methodes Nous avons identifie a l’aide de micropuces les microARNs dont l’expression est modifiee dans des ilots pancreatiques de souris NOD, un modele de souris developpant spontanement un diabete de type I. Les microARNs induits ont ete surexprimes individuellement dans les cellules MIN6 et des cellules β primaires afin de tester leur role dans la synthese et secretion d’insuline et leur effet sur la mort par apoptose. Resultats Nous avons trouve que l’expression des membres de la famille miR-29 (miR-29a/b/c), faisant partie des plus abondants microARNs de la cellule β, augmente avec la mise en place de l’insulite pre-diabetique. Nous avons egalement demontre que ces trois microARNs ont un impacte negatif sur la secretion d’insuline ainsi qu’un effet nefaste sur la survie des cellules β. Une diminution de l’expression de Onecut2, implique dans l’exocytose, ou encore du niveau de phospho-AKT, essentiel pour la survie, dans les cellules transfectees avec miR-29a/b/c pourrait expliquer l’incidence sur les fonctions de la cellule β. Conclusion Nos resultats demontrent que des changements dans l’expression de certains microARNs observes durant l’etablissement du diabete de type I chez la souris NOD, sont responsables de dysfonctionnements au niveau de la cellule β. Par consequent, les microARNs pourraient avoir une part importante a jouer lors du developpement de la maladie.