OBJECTIVEPancreatic 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 METHODSWe 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.RESULTSWe 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.CONCLUSIONSOur 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.

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 ␤-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 ␤-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 ␤-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 ␤-cells is caused by alterations in the level of specific miRNAs. Diabetes 57:2728-2736, 2008

Fine-tuning of insulin secretion from pancreatic b-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 b-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 39-untranslated region of Rab27A mRNA. The effect on the other genes is indirect and linked to changes in mRNA levels. Overexpression 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.

To define the role of the Rab3-interacting molecule RIM in exocytosis we searched for additional binding partners of the protein. We found that the two C 2 domains of RIM display properties analogous to those of the C 2 B domain of synaptotagmin-I. Thus, RIM-C 2 A and RIM-C 2 B bind in a Ca 2؉ -independent manner to ␣1B, the pore-forming subunit of N-type Ca 2؉ channels (EC 50 ‫؍‬ ϳ20 nM). They also weakly interact with the ␣1C but not the ␣1D subunit of L-type Ca 2؉ channels. In addition, the C 2 domains of RIM associate with SNAP-25 and synaptotagmin-I. The binding affinities for these two proteins are 203 and 24 nM, respectively, for RIM-C 2 A and 224 and 16 nM for RIM-C 2 B. The interactions of the C 2 domains of RIM with SNAP-25 and synaptotagmin-I are modulated by Ca 2؉ . Thus, in the presence of Ca 2؉ (EC 50 ‫؍‬ ϳ75 M) the interaction with synaptotagmin-I is increased, whereas SNAP-25 binding is reduced. Synaptotagmin-I binding is abolished by mutations in two positively charged amino acids in the C 2 domains of RIM and by the addition of inositol polyphosphates. We propose that the Rab3 effector RIM is a scaffold protein that participates through its multiple binding partners in the docking and fusion of secretory vesicles at the release sites.Secretion of neurotransmitters, polypeptide hormones, and a variety of other proteins occurs by exocytosis, a multistage process including targeting, docking, and, finally, fusion of secretory vesicles with the plasma membrane. During the last few years, a combination of genetic and biochemical approaches has lead to the identification of several proteins involved in this complex cascade of events. Most of these proteins turned out to be specialized components of the evolutionary conserved machinery that governs intracellular vesicular trafficking in eukaryotic cells (1). Exocytosis was found to necessitate the assembly of a ternary complex between the vesicular SNARE 1 VAMP, associated with the secretory vesicle, and the target SNAREs syntaxin-1 and SNAP-25, localized at the plasma membrane (2). The SNARE complex was initially proposed to ensure the docking of secretory vesicles at the plasma membrane (3). However, it is unlikely that SNARE assembly constitutes the sole determinant for the targeting of secretory vesicles at the release sites because SNARE pairing is rather promiscuous (4, 5), and the localization of syntaxin-1 and SNAP-25 is not restricted to active zones (6). A current hypothesis, supported by biochemical and structural data, proposes that the assembly of the heterotrimeric complex between VAMP, SNAP-25, and syntaxin-1 provides the driving force for membrane fusion (7).In most secretory systems, the exocytotic process is initiated by an increase in the intracellular Ca 2ϩ concentration. In some cells, such as neurons, the elevation of Ca 2ϩ ions is due to opening of voltage-gated calcium channels that are clustered at the release sites, whereas in others, Ca 2ϩ ions are mobilized from intracellular stores. Biochemical and genetic studies indicate...

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.

Granuphilin/Slp-4 is a member of the synaptotagmin-like protein family expressed in pancreatic beta-cells and in the pituitary gland. We show by confocal microscopy that both granuphilin-a and -b colocalize with insulin-containing secretory granules positioned at the periphery of pancreatic beta-cells. Overexpression of granuphilins in insulin-secreting cell lines caused a profound inhibition of stimulus-induced exocytosis. Granuphilins were found to bind to two components of the secretory machinery of pancreatic beta-cells, the small GTP-binding protein Rab3 and the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-binding protein Munc-18. The interaction with Rab3 occurred only with the GTP-bound form of the protein and was prevented by a point mutation in the effector domain of the GTPase. Structure-function studies using granuphilin-b mutants revealed that complete loss of Rab3 binding is associated with a reduction in the capacity to inhibit exocytosis. However, the granuphilin/Rab3 complex alone is not sufficient to mediate the decrease of exocytosis, suggesting the existence of additional binding partners. Taken together, our observations indicate that granuphilins play an important role in pancreatic beta-cell exocytosis. In view of the postulated role of Munc-18 in secretory vesicle docking, our data suggest that granuphilins may also be involved in this process.

Fine-tuning of insulin release from pancreatic β-cells is essential to maintain blood glucose homeostasis. Here, we report that insulin secretion is regulated by a circular RNA containing the lariat sequence of the second intron of the insulin gene. Silencing of this intronic circular RNA in pancreatic islets leads to a decrease in the expression of key components of the secretory machinery of β-cells, resulting in impaired glucose- or KCl-induced insulin release and calcium signaling. The effect of the circular RNA is exerted at the transcriptional level and involves an interaction with the RNA-binding protein TAR DNA-binding protein 43 kDa (TDP-43). The level of this circularized intron is reduced in the islets of rodent diabetes models and of type 2 diabetic patients, possibly explaining their impaired secretory capacity. The study of this and other circular RNAs helps understanding β-cell dysfunction under diabetes conditions, and the etiology of this common metabolic disorder.

Aims/hypothesis Exposure 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. Methods The 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. Results MIN6 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/interpretation Our 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.