The Map3k12 (Dlk)/JNK3 signaling pathway is required for pancreatic beta-cell proliferation during postnatal development.

Unveiling the key pathways underlying postnatal beta-cell proliferation can be instrumental to decipher the mechanisms of beta-cell mass plasticity to increased physiological demand of insulin during weight gain and pregnancy. Using transcriptome and global Serine Threonine Kinase activity (STK) analyses of islets from newborn (10 days old) and adult rats, we found that highly proliferative neonatal rat islet cells display a substantially elevated activity of the mitogen activated protein 3 kinase 12, also called dual leucine zipper-bearing kinase (Dlk). As a key upstream component of the c-Jun amino terminal kinase (Jnk) pathway, Dlk overexpression was associated with increased Jnk3 activity and was mainly localized in the beta-cell cytoplasm. We provide the evidence that Dlk associates with and activates Jnk3, and that this cascade stimulates the expression of Ccnd1 and Ccnd2, two essential cyclins controlling postnatal beta-cell replication. Silencing of Dlk or of Jnk3 in neonatal islet cells dramatically hampered primary beta-cell replication and the expression of the two cyclins. Moreover, the expression of Dlk, Jnk3, Ccnd1 and Ccnd2 was induced in high replicative islet beta cells from ob/ob mice during weight gain, and from pregnant female rats. In human islets from non-diabetic obese individuals, DLK expression was also cytoplasmic and the rise of the mRNA level was associated with an increase of JNK3, CCND1 and CCND2 mRNA levels, when compared to islets from lean and obese patients with diabetes. In conclusion, we find that activation of Jnk3 signalling by Dlk could be a key mechanism for adapting islet beta-cell mass during postnatal development and weight gain.

Mechanisms Underlying the Expansion and Functional Maturation of ß-Cells in Newborns: Impact of the Nutritional Environment.

The functional maturation of insulin-secreting ß-cells is initiated before birth and is completed in early postnatal life. This process has a critical impact on the acquisition of an adequate functional ß-cell mass and on the capacity to meet and adapt to insulin needs later in life. Many cellular pathways playing a role in postnatal ß-cell development have already been identified. However, single-cell transcriptomic and proteomic analyses continue to reveal new players contributing to the acquisition of ß-cell identity. In this review, we provide an updated picture of the mechanisms governing postnatal ß-cell mass expansion and the transition of insulin-secreting cells from an immature to a mature state. We then highlight the contribution of the environment to ß-cell maturation and discuss the adverse impact of an in utero and neonatal environment characterized by calorie and fat overload or by protein deficiency and undernutrition. Inappropriate nutrition early in life constitutes a risk factor for developing diabetes in adulthood and can affect the ß-cells of the offspring over two generations. A better understanding of these events occurring in the neonatal period will help developing better strategies to produce functional ß-cells and to design novel therapeutic approaches for the prevention and treatment of diabetes.

Circular RNAs as Novel Regulators of ß-Cell Functions under Physiological and Pathological Conditions.

Circular RNAs (circRNAs) constitute a large class of non-coding RNAs characterized by a covalently closed circular structure. They originate during mRNA maturation through a modification of the splicing process and, according to the included sequences, are classified as Exonic, Intronic, or Exonic-Intronic. CircRNAs can act by sequestering microRNAs, by regulating the activity of specific proteins, and/or by being translated in functional peptides. There is emerging evidence indicating that dysregulation of circRNA expression is associated with pathological conditions, including cancer, neurological disorders, cardiovascular diseases, and diabetes. The aim of this review is to provide a comprehensive and updated view of the most abundant circRNAs expressed in pancreatic islet cells, some of which originating from key genes controlling the differentiation and the activity of insulin-secreting cells or from diabetes susceptibility genes. We will particularly focus on the role of a group of circRNAs that contribute to the regulation of ß-cell functions and that display altered expression in the islets of rodent diabetes models and of type 2 diabetic patients. We will also provide an outlook of the unanswered questions regarding circRNA biology and discuss the potential role of circRNAs as biomarkers for ß-cell demise and diabetes development.

Crosstalk between Macrophages and Pancreatic ß-Cells in Islet Development, Homeostasis and Disease.

Macrophages are highly heterogeneous and plastic immune cells with peculiar characteristics dependent on their origin and microenvironment. Following pathogen infection or damage, circulating monocytes can be recruited in different tissues where they differentiate into macrophages. Stimuli present in the surrounding milieu induce the polarisation of macrophages towards a pro-inflammatory or anti-inflammatory profile, mediating inflammatory or homeostatic responses, respectively. However, macrophages can also derive from embryonic hematopoietic precursors and reside in specific tissues, actively participating in the development and the homeostasis in physiological conditions. Pancreatic islet resident macrophages are present from the prenatal stages onwards and show specific surface markers and functions. They localise in close proximity to ß-cells, being exquisite sensors of their secretory ability and viability. Over the years, the crucial role of macrophages in ß-cell differentiation and homeostasis has been highlighted. In addition, macrophages are emerging as central players in the initiation of autoimmune insulitis in type 1 diabetes and in the low-grade chronic inflammation characteristic of obesity and type 2 diabetes pathogenesis. The present work reviews the current knowledge in the field, with a particular focus on the mechanisms of communication between ß-cells and macrophages that have been described so far.

Circulating miRNA-150-5p is associated with immune-mediated early ß-cell loss in a humanized mouse model.

BACKGROUND: Aberrant microRNA (miRNA) expression levels are associated with various graft rejections. We used our humanized mouse model with transplanted human islets to identify miRNAs in islet grafts related to xenograft rejection and circulating miRNAs associated with xenograft rejection-mediated ß-cell loss. METHODS: Diabetic immunodeficient NOD.scid mice were transplanted with human islets and subsequently achieved stable normoglycemia. Lymphocytes from NOD mice were then adoptively transferred to the humanized mice to induce human ß-cell destruction. Islet graft and plasma were collected immediately once blood glucose reached >200 mg/dL. miRNAs in the islet grafts and in the plasma with or without adoptive lymphocyte transfer (ALT) were measured using NanoString nCounter® miRNA Expression Assay and qPCR. RESULTS: A set of immune-related miRNAs was significantly increased in human islet grafts of ALT-treated mice compared to control mice. Of these miRNAs, miR-150-5p was significantly increased in the circulation of ALT-treated mice at tissue collection and the increase was a result of immune activation rather than simply the presence of lymphocytes in circulation. Furthermore, miR-150-5p was significantly increased in human islet graft and circulation prior to the development of hyperglycemia in the ALT-treated mice. CONCLUSIONS: Our data demonstrated that immune-related miRNAs are associated with human islet xenograft rejection in mice. miR-150-5p is increased in human islet graft and in the circulation during islet xenograft rejection and ß-cell destruction prior to hyperglycemia and may be an early biomarker for islet xenograft rejection.

PIWI-interacting RNAs as novel regulators of pancreatic beta cell function.

AIMS/HYPOTHESIS: P-element induced Wimpy testis (PIWI)-interacting RNAs (piRNAs) are small non-coding RNAs that interact with PIWI proteins and guide them to silence transposable elements. They are abundantly expressed in germline cells and play key roles in spermatogenesis. There is mounting evidence that piRNAs are also present in somatic cells, where they may accomplish additional regulatory tasks. The aim of this study was to identify the piRNAs expressed in pancreatic islets and to determine whether they are involved in the control of beta cell activities. METHODS: piRNA profiling of rat pancreatic islets was performed by microarray analysis. The functions of piRNAs were investigated by silencing the two main Piwi genes or by modulating the level of selected piRNAs in islet cells. RESULTS: We detected about 18,000 piRNAs in rat pancreatic islets, many of which were differentially expressed throughout islet postnatal development. Moreover, we identified changes in the level of several piRNAs in the islets of Goto-Kakizaki rats, a well-established animal model of type 2 diabetes. Silencing of Piwil2 or Piwil4 genes in adult rat islets caused a reduction in the level of several piRNAs and resulted in defective insulin secretion and increased resistance of the cells to cytokine-induced cell death. Furthermore, overexpression in the islets of control animals of two piRNAs that are upregulated in diabetic rats led to a selective defect in glucose-induced insulin release. CONCLUSIONS/INTERPRETATION: Our results provide evidence for a role of PIWI proteins and their associated piRNAs in the control of beta cell functions, and suggest a possible involvement in the development of type 2 diabetes. DATA AVAILABILITY: Data have been deposited in Gene Expression Omnibus repository under the accession number GSE93792. Data can be accessed via the following link: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=ojklueugdzehpkv&acc=GSE93792.

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.

New emerging tasks for microRNAs in the control of ß-cell activities.

MicroRNAs are key regulators of ß-cell physiology. They participate to the differentiation of insulin-producing cells and are instrumental for the acquisition of their unique secretory properties. Moreover, they contribute to the adaptation of ß-cells to conditions of increased insulin demand and, if expressed at inappropriate levels, certain microRNAs cause ß-cell dysfunction and promote the development of different forms of diabetes mellitus. While these functions are increasingly better understood, additional tasks for these small non-coding RNAs have been recently unveiled. Thus, microRNAs are emerging as signaling molecules of a novel exosome-mediated cell-to-cell communication mode permitting a coordinated response of the ß-cells to inflammatory conditions and to modifications in the insulin demand. These discoveries raise a number of important issues that once addressed promise to shed new light on the molecular mechanism governing the functions of the ß-cells under normal and disease states. This article is part of a Special Issue entitled: MicroRNAs and lipid/energy metabolism and related diseases edited by Carlos Fernández-Hernando and Yajaira Suárez.

Exosomes as new players in metabolic organ cross-talk.

Blood glucose homeostasis requires a constant communication between insulin-secreting and insulin-sensitive cells. A wide variety of circulating factors, including hormones, cytokines and chemokines work together to orchestrate the systemic response of metabolic organs to changes in the nutritional state. Failure in the coordination between these organs can lead to a rise in blood glucose levels and to the appearance of metabolic disorders such as diabetes mellitus. Exosomes are small extracellular vesicles (EVs) that are produced via the endosomal pathway and are released from the cells upon fusion of multivesicular bodies with the plasma membrane. There is emerging evidence indicating that these EVs play a central role in cell-to-cell communication. The interest in exosomes exploded when they were found to transport bioactive proteins, messenger RNA (mRNAs) and microRNA (miRNAs) that can be transferred in active form to adjacent cells or to distant organs. In this review, we will first outline the mechanisms governing the biogenesis, the cargo upload and the release of exosomes by donor cells as well as the uptake by recipient cells. We will then summarize the studies that support the novel concept that miRNAs and other exosomal cargo components are new important vehicles for metabolic organ cross-talk.

Role of microRNAs in the age-associated decline of pancreatic beta cell function in rat islets.

AIMS/HYPOTHESIS: Ageing can lead to reduced insulin sensitivity and loss of pancreatic beta cell function, predisposing individuals to the development of diabetes. The aim of this study was to assess the contribution of microRNAs (miRNAs) to age-associated beta cell dysfunction. METHODS: The global mRNA and miRNA profiles of 3- and 12-month-old rat islets were collected by microarray. The functional impact of age-associated differences in miRNA expression was investigated by mimicking the observed changes in primary beta cells from young animals. RESULTS: Beta cells from 12-month-old rats retained normal insulin content and secretion, but failed to proliferate in response to mitotic stimuli. The islets of these animals displayed modifications at the level of several miRNAs, including upregulation of miR-34a, miR-124a and miR-383, and downregulation of miR-130b and miR-181a. Computational analysis of the transcriptomic modifications observed in the islets of 12-month-old rats revealed that the differentially expressed genes were enriched for miR-34a and miR-181a targets. Indeed, the induction of miR-34a and reduction of miR-181a in the islets of young animals mimicked the impaired beta cell proliferation observed in old animals. mRNA coding for alpha-type platelet-derived growth factor receptor, which is critical for compensatory beta cell mass expansion, is directly inhibited by miR34a and is likely to be at least partly responsible for the effects of this miRNA. CONCLUSIONS/INTERPRETATION: Changes in the level of specific miRNAs that occur during ageing affect the proliferative capacity of beta cells. This might reduce their ability to expand under conditions of increased insulin demand, favouring the development of type 2 diabetes.

Exosome-like vesicles released from lipid-induced insulin-resistant muscles modulate gene expression and proliferation of beta recipient cells in mice.

AIMS/HYPOTHESIS: The crosstalk between skeletal muscle (SkM) and beta cells plays a role in diabetes aetiology. In this study, we have investigated whether SkM-released exosome-like vesicles (ELVs) can be taken up by pancreatic beta cells and can deliver functional cargoes. METHODS: Mice were fed for 16 weeks with standard chow diet (SCD) or with standard diet enriched with 20% palmitate (HPD) and ELVs were purified from quadriceps muscle. Fluorescent ELVs from HPD or SCD quadriceps were injected i.v. or intramuscularly (i.m.) into mice to determine their biodistributions. Micro (mi)RNA quantification in ELVs was determined using quantitative real-time RT-PCR (qRT-PCR)-based TaqMan low-density arrays. Microarray analyses were performed to determine whether standard diet ELVs (SD-ELVs) and high palmitate diet ELVs (HPD-ELVs) induced specific transcriptional signatures in MIN6B1 cells. RESULTS: In vivo, muscle ELVs were taken up by pancreas, 24 h post-injection. In vitro, both SD-ELVs and HPD-ELVs transferred proteins and miRNAs to MIN6B1 cells and modulated gene expressions whereas only HPD-ELVs induced proliferation of MIN6B1 cells and isolated islets. Bioinformatic analyses suggested that transferred HPD-ELV miRNAs may participate in these effects. To validate this, we demonstrated that miR-16, which is overexpressed in HPD-ELVs, was transferred to MIN6B1 cells and regulated Ptch1, involved in pancreas development. In vivo, islets from HPD mice showed increased size and altered expression of genes involved in development, including Ptch1, suggesting that the effect of palm oil on islet size in vivo was reproduced in vitro by treating beta cells with HPD-ELVs. CONCLUSIONS/INTERPRETATION: Our data suggest that muscle ELVs might have an endocrine effect and could participate in adaptations in beta cell mass during insulin resistance.

Role of islet microRNAs in diabetes: which model for which question?

MicroRNAs are important regulators of gene expression. The vast majority of the cells in our body rely on hundreds of these tiny non-coding RNA molecules to precisely adjust their protein repertoire and faithfully accomplish their tasks. Indeed, alterations in the microRNA profile can lead to cellular dysfunction that favours the appearance of several diseases. A specific set of microRNAs plays a crucial role in pancreatic beta cell differentiation and is essential for the fine-tuning of insulin secretion and for compensatory beta cell mass expansion in response to insulin resistance. Recently, several independent studies reported alterations in microRNA levels in the islets of animal models of diabetes and in islets isolated from diabetic patients. Surprisingly, many of the changes in microRNA expression observed in animal models of diabetes were not detected in the islets of diabetic patients and vice versa. These findings are unlikely to merely reflect species differences because microRNAs are highly conserved in mammals. These puzzling results are most probably explained by fundamental differences in the experimental approaches which selectively highlight the microRNAs directly contributing to diabetes development, the microRNAs predisposing individuals to the disease or the microRNAs displaying expression changes subsequent to the development of diabetes. In this review we will highlight the suitability of the different models for addressing each of these questions and propose future strategies that should allow us to obtain a better understanding of the contribution of microRNAs to the development of diabetes mellitus in humans.

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.

Horizontal transfer of exosomal microRNAs transduce apoptotic signals between pancreatic beta-cells.

BACKGROUND: Diabetes mellitus is a common metabolic disorder characterized by dysfunction of insulin-secreting pancreatic beta-cells. MicroRNAs are important regulators of beta-cell activities. These non-coding RNAs have recently been discovered to exert their effects not only inside the cell producing them but, upon exosome-mediated transfer, also in other recipient cells. This novel communication mode remains unexplored in pancreatic beta-cells. In the present study, the microRNA content of exosomes released by beta-cells in physiological and physiopathological conditions was analyzed and the biological impact of their transfer to recipient cells investigated. RESULTS: Exosomes were isolated from the culture media of MIN6B1 and INS-1 derived 832/13 beta-cell lines and from mice, rat or human islets. Global profiling revealed that the microRNAs released in MIN6B1 exosomes do not simply reflect the content of the cells of origin. Indeed, while a subset of microRNAs was preferentially released in exosomes others were selectively retained in the cells. Moreover, exposure of MIN6B1 cells to inflammatory cytokines changed the release of several microRNAs. The dynamics of microRNA secretion and their potential transfer to recipient cells were next investigated. As a proof-of-concept, we demonstrate that if cel-miR-238, a C. Elegans microRNA not present in mammalian cells, is expressed in MIN6B1 cells a fraction of it is released in exosomes and is transferred to recipient beta-cells. Furthermore, incubation of untreated MIN6B1 or mice islet cells in the presence of microRNA-containing exosomes isolated from the culture media of cytokine-treated MIN6B1 cells triggers apoptosis of recipient cells. In contrast, exosomes originating from cells not exposed to cytokines have no impact on cell survival. Apoptosis induced by exosomes produced by cytokine-treated cells was prevented by down-regulation of the microRNA-mediating silencing protein Ago2 in recipient cells, suggesting that the effect is mediated by the non-coding RNAs. CONCLUSIONS: Taken together, our results suggest that beta-cells secrete microRNAs that can be transferred to neighboring beta-cells. Exposure of donor cells to pathophysiological conditions commonly associated with diabetes modifies the release of microRNAs and affects survival of recipient beta-cells. Our results support the concept that exosomal microRNAs transfer constitutes a novel cell-to-cell communication mechanism regulating the activity of pancreatic beta-cells.

Molecular mechanism of myosin Va recruitment to dense core secretory granules.

The brain-spliced isoform of Myosin Va (BR-MyoVa) plays an important role in the transport of dense core secretory granules (SGs) to the plasma membrane in hormone and neuropeptide-producing cells. The molecular composition of the protein complex that recruits BR-MyoVa to SGs and regulates its function has not been identified to date. We have identified interaction between SG-associated proteins granuphilin-a/b (Gran-a/b), BR-MyoVa and Rab27a, a member of the Rab family of GTPases. Gran-a/b-BR-MyoVa interaction is direct, involves regions downstream of the Rab27-binding domain, and the C-terminal part of Gran-a determines exon specificity. MyoVa and Gran-a/b are partially colocalised on SGs and disruption of Gran-a/b-BR-MyoVa binding results in a perinuclear accumulation of SGs which augments nutrient-stimulated hormone secretion in pancreatic beta-cells. These results indicate the existence of at least another binding partner of BR-MyoVa that was identified as rabphilin-3A (Rph-3A). BR-MyoVa-Rph-3A interaction is also direct and enhanced when secretion is activated. The BR-MyoVa-Rph-3A and BR-MyoVa-Gran-a/b complexes are linked to a different subset of SGs, and simultaneous inhibition of these complexes nearly completely blocks stimulated hormone release. This study demonstrates that multiple binding partners of BR-MyoVa regulate SG transport, and this molecular mechanism is universally used by neuronal, endocrine and neuroendocrine cells.

The mitochondrial tRNA-derived fragment, mt-tRF-LeuTAA, couples mitochondrial metabolism to insulin secretion.

OBJECTIVE: The contribution of the mitochondrial electron transfer system to insulin secretion involves more than just energy provision. We identified a small RNA fragment (mt-tRF-LeuTAA) derived from the cleavage of a mitochondrially-encoded tRNA that is conserved between mice and humans. The role of mitochondrially-encoded tRNA-derived fragments remains unknown. This study aimed to characterize the impact of mt-tRF-LeuTAA, on mitochondrial metabolism and pancreatic islet functions. METHODS: We used antisense oligonucleotides to reduce mt-tRF-LeuTAA levels in primary rat and human islet cells, as well as in insulin-secreting cell lines. We performed a joint transcriptome and proteome analysis upon mt-tRF-LeuTAA inhibition. Additionally, we employed pull-down assays followed by mass spectrometry to identify direct interactors of the fragment. Finally, we characterized the impact of mt-tRF-LeuTAA silencing on the coupling between mitochondrial metabolism and insulin secretion using high-resolution respirometry and insulin secretion assays. RESULTS: Our study unveils a modulation of mt-tRF-LeuTAA levels in pancreatic islets in different Type 2 diabetes models and in response to changes in nutritional status. The level of the fragment is finely tuned by the mechanistic target of rapamycin complex 1. Located within mitochondria, mt-tRF-LeuTAA interacts with core subunits and assembly factors of respiratory complexes of the electron transfer system. Silencing of mt-tRF-LeuTAA in islet cells limits the inner mitochondrial membrane potential and impairs mitochondrial oxidative phosphorylation, predominantly by affecting the Succinate (via Complex II)-linked electron transfer pathway. Lowering mt-tRF-LeuTAA impairs insulin secretion of rat and human pancreatic ß-cells. CONCLUSIONS: Our findings indicate that mt-tRF-LeuTAA interacts with electron transfer system complexes and is a pivotal regulator of mitochondrial oxidative phosphorylation and its coupling to insulin secretion.

Identification of particular groups of microRNAs that positively or negatively impact on beta cell function in obese models of type 2 diabetes.

AIMS/HYPOTHESIS: MicroRNAs are key regulators of gene expression involved in health and disease. The goal of our study was to investigate the global changes in beta cell microRNA expression occurring in two models of obesity-associated type 2 diabetes and to assess their potential contribution to the development of the disease. METHODS: MicroRNA profiling of pancreatic islets isolated from prediabetic and diabetic db/db mice and from mice fed a high-fat diet was performed by microarray. The functional impact of the changes in microRNA expression was assessed by reproducing them in vitro in primary rat and human beta cells. RESULTS: MicroRNAs differentially expressed in both models of obesity-associated type 2 diabetes fall into two distinct categories. A group including miR-132, miR-184 and miR-338-3p displays expression changes occurring long before the onset of diabetes. Functional studies indicate that these expression changes have positive effects on beta cell activities and mass. In contrast, modifications in the levels of miR-34a, miR-146a, miR-199a-3p, miR-203, miR-210 and miR-383 primarily occur in diabetic mice and result in increased beta cell apoptosis. These results indicate that obesity and insulin resistance trigger adaptations in the levels of particular microRNAs to allow sustained beta cell function, and that additional microRNA deregulation negatively impacting on insulin-secreting cells may cause beta cell demise and diabetes manifestation. CONCLUSIONS/INTERPRETATION: We propose that maintenance of blood glucose homeostasis or progression toward glucose intolerance and type 2 diabetes may be determined by the balance between expression changes of particular microRNAs.

Overexpression of miR-297b-5p in Mouse Insulin-Secreting Cells Promotes Metformin-Mediated Protection Against Stearic Acid-Induced Senescence by Targeting Igf1r.

BACKGROUND: A long-term consumption of saturated fat significantly increases the concentration of saturated fatty acids in serum, which accelerates the appearance of senescence markers in ß-cells and leads to their dysfunction. An understanding of the mechanisms underlying ß-cell senescence induced by stearic acid and the exploration of effective agents preventing it remains largely unclear. Here, we aimed to investigate the protective effect of metformin against stearic acid-treated ß-cell senescence and to assess the involvement of miR-297b-5p in this process. METHODS: To identify senescence, we measured senescence-associated ß-galactosidase activity and the expression of senescence-related genes. Gain and loss of function approaches were applied to explore the role of miR-297b-5p in stearic acid-induced ß-cell senescence. Bioinformatics analysis and a luciferase activity assay were used to predict the downstream targets of miR-297b-5p. RESULTS: Stearic acid markedly induced senescence and suppressed miR-297b-5p expression in mouse ß-TC6 cells, which were significantly alleviated by metformin. After transfection of miR-297b-5p mimics, stearic acid-evoked ß-cell senescence was remarkably prevented. Insulin-like growth factor-1 receptor was identified as a direct target of miR-297b-5p. Inhibition of the insulin-like growth factor-1 receptor prevented stearic acid-induced ß-cell senescence and dysfunction. Moreover, metformin alleviates the impairment of the miR-297b-5p inhibitor in ß-TC6 cells. Additionally, long-term consumption of a high-stearic-acid diet significantly increased senescence and reduced miR-297b-5p expression in mouse islets. CONCLUSIONS: These findings imply that metformin alleviates ß-cell senescence by stearic acid through upregulating miR-297b-5p to suppress insulin-like growth factor-1 receptor expression, thereby providing a potential target to not only prevent high fat-diet-induced ß-cell dysfunction but also for metformin therapy in type 2 diabetes.

Regulation of insulin secretion by phosphatidylinositol-4,5-bisphosphate.

The role of PIP(2) in pancreatic beta cell function was examined here using the beta cell line MIN6B1. Blocking PIP(2) with PH-PLC-GFP or PIP5KIgamma RNAi did not impact on glucose-stimulated secretion although susceptibility to apoptosis was increased. Over-expression of PIP5KIgamma improved cell survival and inhibited secretion with accumulation of endocytic vacuoles containing F-actin, PIP(2), transferrin receptor, caveolin 1, Arf6 and the insulin granule membrane protein phogrin but not insulin. Expression of constitutively active Arf6 Q67L also resulted in vacuole formation and inhibition of secretion, which was reversed by PH-PLC-GFP co-expression. PIP(2) co-localized with gelsolin and F-actin, and gelsolin co-expression partially reversed the secretory defect of PIP5KIgamma-over-expressing cells. RhoA/ROCK inhibition increased actin depolymerization and secretion, which was prevented by over-expressing PIP5KIgamma, while blocking PIP(2) reduced constitutively active RhoA V14-induced F-actin polymerization. In conclusion, although PIP(2) plays a pro-survival role in MIN6B1 cells, excessive PIP(2) production because of PIP5KIgamma over-expression inhibits secretion because of both a defective Arf6/PIP5KIgamma-dependent endocytic recycling of secretory membrane and secretory membrane components such as phogrin and the RhoA/ROCK/PIP5KIgamma-dependent perturbation of F-actin cytoskeleton remodelling.