A circular RNA generated from an intron of the insulin gene controls insulin secretion.

Fine-tuning of insulin release from pancreatic ß-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 ß-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 ß-cell dysfunction under diabetes conditions, and the etiology of this common metabolic disorder.

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

OBJECTIVE: Non-coding RNAs constitute a major fraction of the ß-cell transcriptome. While the involvement of microRNAs is well established, the contribution of long non-coding RNAs (lncRNAs) in the regulation of ß-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 ß-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 ß-cells, ßlinc2, and ßlinc3, 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 ßlinc3 was altered in the islets of type 2 diabetic patients and was associated to the BMI of the donors. Modulation of the level of ßlinc2 and ßlinc3 by overexpression or downregulation in MIN6 and mouse islet cells did not affect insulin secretion but increased ß-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 ß-cell failure during the development of the disease.

MicroRNAs modulate core-clock gene expression in pancreatic islets during early postnatal life in rats.

AIMS/HYPOTHESIS: Evidence continues to emerge detailing a fine-tuning of the regulation of metabolic processes and energy homeostasis by cell-autonomous circadian clocks. Pancreatic beta cell functional maturation occurs after birth and implies transcriptional changes triggered by a shift in the nutritional supply that occurs at weaning, enabling the adaptation of insulin secretion. So far, the developmental timing and exact mechanisms involved in the initiation of the circadian clock in the growing pancreatic islets have never been addressed. METHODS: Circadian gene expression was measured by quantitative RT-PCR in islets of rats at different postnatal ages up to 3 months, and by in vitro bioluminescence recording in newborn (10-day-old) and adult (3-month-old) islets. The effect of the microRNAs miR-17-5p and miR-29b-3p on the expression of target circadian genes was assessed in newborn rat islets transfected with microRNA antisense or mimic oligonucleotides, and luciferase reporter assays were performed on the rat insulin-secreting cell line INS832/13 to determine a direct effect. The global regulatory network between microRNAs and circadian genes was computationally predicted. RESULTS: We found up to a sixfold-change in the 24 h transcriptional oscillations and overall expression of Clock, Npas2, Bmal1, Bmal2, Rev-erbα, Per1, Per2, Per3 and Cry2 between newborn and adult rat islets. Synchronisation of the clock machinery in cultured islet cells revealed a delayed cell-autonomous rhythmicity of about 1.5 h in newborn compared with adult rats. Computational predictions unveiled the existence of a complex regulatory network linking over 40 microRNAs displaying modifications in their expression profiles during postnatal beta cell maturation and key core-clock genes. In agreement with these computational predictions, we demonstrated that miR-17-5p and miR-29b-3p directly regulated circadian gene expression in the maturing islet cells of 10-day-old rats. CONCLUSIONS/INTERPRETATION: These data show that the circadian clock is not fully operational in newborn islets and that microRNAs potently contribute to its regulation during postnatal beta cell maturation. Defects in this process may have long-term consequences on circadian physiology and pancreatic islet function, favouring the manifestation of metabolic diseases such as diabetes.

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

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.

Changes in microRNA expression contribute to pancreatic ß-cell dysfunction in prediabetic NOD mice.

During the initial phases of type 1 diabetes, pancreatic islets are invaded by immune cells, exposing ß-cells to proinflammatory cytokines. This unfavorable environment results in gene expression modifications leading to loss of ß-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 ß-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 ß-cell dysfunction occurring during the initial phases of type 1 diabetes.

Involvement of microRNAs in the cytotoxic effects exerted by proinflammatory cytokines on pancreatic beta-cells.

OBJECTIVE: Pancreatic beta-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 beta-cell cytotoxicity. RESEARCH DESIGN AND METHODS: We used global microarray profiling and real-time PCR analysis to detect changes in microRNA expression in beta-cells exposed to cytokines and in islets of pre-diabetic NOD mice. We assessed the involvement of the microRNAs affected in cytokine-mediated beta-cell failure by modifying their expression in insulin-secreting MIN6 cells. RESULTS: We found that IL-1beta and TNF-alpha 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-1beta 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 beta-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.

Alterations in microRNA expression contribute to fatty acid-induced pancreatic beta-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 beta-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 beta-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 beta-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 beta-cells is caused by alterations in the level of specific miRNAs.

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

Fine-tuning of insulin secretion from pancreatic beta-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 beta-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.

Pancreatic beta-cell protein granuphilin binds Rab3 and Munc-18 and controls exocytosis.

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.

The death domain of Rab3 guanine nucleotide exchange protein in GDP/GTP exchange activity in living cells.

Rab3 GTPases regulate exocytosis of neurons, endocrine and exocrine cells. In the present paper, we report a system to measure the guanine nucleotide status of Rab3 proteins in living cells. The assay is based on the ability of the Rab3 interacting molecule RIM to extract selectively the GTP-bound form of Rab3. Using this system, we found that approx. 20% of wild-type Rab3A, -B, -C or -D transfected in the insulin-secreting cell line HIT-T15 is in the GTP-bound conformation. The pool of activated Rab3 is decreased under conditions that stimulate exocytosis or by co-expression of the Rab3 GTPase-activating protein. In contrast, co-expression of Mss4 or Rab3-GEP (guanine nucleotide exchange protein) increases by approx. 3-fold the GTP-bound pool of Rab3 isoforms. Rab3-GEP is very similar to MADD, a death domain-containing protein that associates with the type 1 tumour necrosis factor receptor. We observed that the death domain of Rab3-GEP is involved in intramolecular interactions and that deletions or mutations that affect this domain of the protein impair the nucleotide exchange activity towards Rab3. We propose that the death domain of Rab3-GEP acts as a molecular switch and co-ordinates multiple functions of the protein by exchanging its binding partners.