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SummaryThe 3’ untranslated region (3’UTR) plays a crucial role in determining mRNA stability, localisation, translation and degradation. Cap analysis gene expression (CAGE), a method for the detection of capped 5’ ends of mRNAs, additionally reveals a large number of apparently 5’ capped RNAs derived from 3’UTRs. Here we provide the first direct evidence that these 3’UTR-derived RNAs are indeed capped and often more abundant than the corresponding full-length mRNAs. By using a combination of AGO2 enhanced individual nucleotide resolution UV crosslinking and immunoprecipitation (eiCLIP) and CAGE following siRNA knockdowns, we find that these 3’UTR-derived RNAs likely originate from AGO2-mediated cleavage, and most often occur at locations with potential to form RNA-G-quadruplexes and are enriched by RNA-binding protein UPF1. High-resolution imaging and long-read sequencing analysis validates several 3’UTR-derived RNAs, demonstrates their abundance and shows that they tend not to co-localise with the parental mRNAs. Taken together, we provide new insights into the origin and abundance of 3’UTR-derived RNAs, show the utility of CAGE-seq for their quantitative detection, and provide a rich dataset for exploring new biology of a poorly understood new class of RNAs.

Aims/hypothesis Although targeted in extrapancreatic tissues by several drugs used to treat type 2 diabetes, the role of AMP-activated protein kinase (AMPK) in the control of insulin secretion is still debatable. Previous studies have used pharmacological activators of limited selectivity and specificity, and none has examined in primary pancreatic beta cells the actions of the latest generation of highly potent and specific activators that act via the allosteric drug and metabolite (ADaM) site. Methods AMPK was activated acutely in islets isolated from C57BL6/J mice, and in an EndoC-βH3 cell line, using three structurally distinct ADaM site activators (991, PF-06409577 and RA089), with varying selectivity for β1- vs β2-containing complexes. Mouse lines expressing a gain-of-function mutation in the γ1 AMPK subunit (D316a) were generated to examine the effects of chronic AMPK stimulation in the whole body, or selectively in the beta cell. Results Acute (1.5 h) treatment of wild-type mouse islets with 991, PF-06409577 or RA089 robustly stimulated insulin secretion at high glucose concentrations (ppp2+ dynamics in response to 991 (AUC, ppppp Conclusions/interpretation AMPK activation exerts complex, time-dependent effects on insulin secretion. These observations should inform the design and future clinical use of AMPK modulators. Graphical abstract

Aim: We previously discovered that 14-3-3-zeta (14-3-3ζ) was essential for adipocyte differentiation. Although 14-3-3ζ scaffold is not a transcription factor (TF) , it undergoes nuclear translocation during adipogenesis. Considering its diverse interactome, we hypothesized that 14-3-3ζ anchors, nucleates and regulates essential transcriptional complexes required for adipogenesis. Methods: 3T3-L1 pre-adipocytes were modified by CRISPR-Cas9 to express a TAP (Flag-HA) -tagged 14-3-3ζ protein. At 48 first hours of adipogenesis, nuclear TAP-14-3-3-ζ complexes were purified to identify and annotate the 14-3-3ζ interactome by MS and Gene Ontology. Highly enriched proteins functions were assessed for their roles in adipogenesis using pharmacological inhibitors or siRNA. In parallel, chromatin accessibility was assessed by ATAC-Seq from control or 14-3-3ζ-depleted 3T3-L1 cells during early adipogenesis. Results: We found that the 14-3-3ζ nuclear interactome during the early stages of adipogenesis was significantly enriched with 133 proteins, including CEBP-β, an early adipogenic TF. According to GO analysis, proteins within the 14-3-3ζ interactome were primarily associated (FDR ≤ 0.01) with histone H2B ubiquitination, DNA unwinding for replication, DNA hypermethylation and ribosome assembly. Pharmacological inhibition or depletion by siRNA of these proteins significantly dampened adipocyte differentiation. Principal component analysis of ATAC-seq results revealed that samples from 14-3-3ζ-depleted cells differed from controls in term of whole chromatin accessibility, and DESeq2 identified nearly 3400 (p Conclusion: These results reveal that via critical protein interactions, 14-3-3ζ has essential roles in chromatin accessibility, DNA replication, and mRNA translation, which are crucial events for the early adipogenesis. Disclosure S.A.Rial: None. A.Alkhoury: None. G.Lavoie: None. P.Roux: None. T.M.Durcan: None. A.Martinez-sanchez: None. G.E.Lim: None. Funding Natural Sciences and Engineering Research Council of Canada (NSERC) ; Canada Research Chairs (CRC) ; Fonds de recherche du Québec-Nature and Technologies (FRQNT)

Pulsatile increases in Ca2+ within beta cells underlie the stimulation of insulin secretion at elevated glucose concentrations. Recent data obtained by imaging these transients in intact islets have revealed the existence of subpopulations of beta cells (“leaders”) which initiate Ca2+ waves across the syncytium and are critical for these dynamics. Whether leader cells possess unique transcriptional features is unknown. By combining Ca2+ imaging with cell labelling and isolation, we sought to address this question. Method: Islets were isolated from C57BL6/J mice expressing the Ca2+ probe GCaMP6f selectively in beta cells. After infection with an adenovirus expressing photo-activable- (PA) mCherry, Ca2+ imaging was performed at 11mM glucose at 5 Hz. Leader (or follower) cells were identified and photopainted by PA-mCherry activation at 405nm. After islet dispersal, cell sorting was used to isolate single cells. cDNA libraries were obtained using NEBNext Single cell kit and used for sequencing (Illumina HiSeq 4000) . Reads were aligned to the mmtranscriptome with Salmon v1.3 and differential expression analysis performed with DESeq2. Differentially expressed genes were subjected to Gene Ontology analysis. Results: cDNA was obtained from 13 leader cells, identified as those exhibiting the first Ca2+ signal increase during Ca2+ waves, and 9 follower cells. Of 96 differentially expressed genes, the most strongly affected included cell adhesion proteins (Cdh22; Scarf2) and the calcium sensor Mctp1. Other genes identified were involved in mitochondrial Ca2+ uptake (Mcub) , cytochrome assembly (Tmem177) and type 2 diabetes risk (Tcf7l2) . GO analysis identified 9 enriched functional clusters, including protein ubiquitination and metal ion binding. Conclusion: Leader cells possess a stably altered transcriptome, and the affected genes include several which may regulate cell adhesion. The extent to which the identified genes reflect the contribution of these cells to Ca2+ wave initiation requires further study. Disclosure P.L.Chabosseau: n/a. S.Yong: None. L.Lopez: None. I.Leclerc: None. Y.Ali: n/a. A.Martinez-sanchez: None. G.A.Rutter: Advisory Panel; Sun Pharmaceutical Industries Ltd., Research Support; Sun Pharmaceutical Industries Ltd.

Aims/hypothesis Variants close to the VPS13C/C2CD4A/C2CD4B locus are associated with altered risk of type 2 diabetes in genome-wide association studies. While previous functional work has suggested roles for VPS13C and C2CD4A in disease development, none has explored the role of C2CD4B. Methods CRISPR/Cas9-induced global C2cd4b-knockout mice and zebrafish larvae with c2cd4a deletion were used to study the role of this gene in glucose homeostasis. C2 calcium dependent domain containing protein (C2CD)4A and C2CD4B constructs tagged with FLAG or green fluorescent protein were generated to investigate subcellular dynamics using confocal or near-field microscopy and to identify interacting partners by mass spectrometry. Results Systemic inactivation of C2cd4b in mice led to marked, but highly sexually dimorphic changes in body weight and glucose homeostasis. Female C2cd4b mice displayed unchanged body weight compared with control littermates, but abnormal glucose tolerance (AUC, p = 0.01) and defective in vivo, but not in vitro, insulin secretion (p = 0.02). This was associated with a marked decrease in follicle-stimulating hormone levels as compared with wild-type (WT) littermates (p = 0.003). In sharp contrast, male C2cd4b null mice displayed essentially normal glucose tolerance but an increase in body weight (p p = 0.003) after maintenance on a high-fat and -sucrose diet vs WT littermates. No metabolic disturbances were observed after global inactivation of C2cd4a in mice, or in pancreatic beta cell function at larval stages in C2cd4a null zebrafish. Fasting blood glucose levels were also unaltered in adult C2cd4a-null fish. C2CD4B and C2CD4A were partially localised to the plasma membrane, with the latter under the control of intracellular Ca2+. Binding partners for both included secretory-granule-localised PTPRN2/phogrin. Conclusions/interpretation Our studies suggest that C2cd4b may act centrally in the pituitary to influence sex-dependent circuits that control pancreatic beta cell function and glucose tolerance in rodents. However, the absence of sexual dimorphism in the impact of diabetes risk variants argues for additional roles for C2CD4A or VPS13C in the control of glucose homeostasis in humans. Data availability The datasets generated and/or analysed during the current study are available in the Biorxiv repository (www.biorxiv.org/content/10.1101/2020.05.18.099200v1). RNA-Seq (GSE152576) and proteomics (PXD021597) data have been deposited to GEO (www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE152576) and ProteomeXchange (www.ebi.ac.uk/pride/archive/projects/PXD021597) repositories, respectively. Graphical abstract

All forms of diabetes mellitus involve the loss or dysfunction of pancreatic beta cells, with the former predominating in type 1 diabetes and the latter in type 2 diabetes. Deeper understanding of the coupling mechanisms that link glucose metabolism in these cells to the control of insulin secretion is therefore likely to be essential to develop new therapies. Beta cells display a remarkable metabolic specialisation, expressing high levels of metabolic sensing enzymes, including the glucose transporter GLUT2 (encoded by SLC2A2) and glucokinase (encoded by GCK). Genetic evidence flowing from both monogenic forms of diabetes and genome-wide association studies for the more common type 2 diabetes, supports the importance for normal glucose-stimulated insulin secretion of metabolic signalling via altered ATP generation, while also highlighting unsuspected roles for Zn2+ storage, intracellular lipid transfer and other processes. Intriguingly, genes involved in non-oxidative metabolic fates of the sugar, such as those for lactate dehydrogenase (LDHA) and monocarboxylate transporter-1 ([MCT-1] SLC16A1), as well as the acyl-CoA thioesterase (ACOT7) and others, are selectively repressed (‘disallowed’) in beta cells. Furthermore, mutations in genes critical for mitochondrial oxidative metabolism, such as TRL-CAG1–7 encoding tRNALeu, are linked to maternally inherited forms of diabetes. Correspondingly, impaired Ca2+ uptake into mitochondria, or collapse of a normally interconnected mitochondrial network, are associated with defective insulin secretion. Here, we suggest that altered mitochondrial metabolism may also impair beta cell–beta cell communication. Thus, we argue that defective oxidative glucose metabolism is central to beta cell failure in diabetes, acting both at the level of single beta cells and potentially across the whole islet to impair insulin secretion.

AIMS: Spatially-organized increases in cytosolic Ca2+ within pancreatic beta cells in the pancreatic islet underlie the stimulation of insulin secretion by high glucose. Recent data have revealed the existence of subpopulations of beta cells including "leaders" which initiate Ca2+ waves. Whether leader cells possess unique molecular features, or localisation, is unknown. MAIN METHODS: High speed confocal Ca2+ imaging was used to identify leader cells and connectivity analysis, running under MATLAB and Python, to identify highly connected "hub" cells. To explore transcriptomic differences between beta cell sub-groups, individual leaders or followers were labelled by photo-activation of the cryptic fluorescent protein PA-mCherry and subjected to single cell RNA sequencing ("Flash-Seq"). KEY FINDINGS: Distinct Ca2+ wave types were identified in individual islets, with leader cells present in 73 % (28 of 38 islets imaged). Scale-free, power law-adherent behaviour was also observed in 29 % of islets, though "hub" cells in these islets did not overlap with leaders. Transcripts differentially expressed (295; padj

Pancreatic β-cells within the islets of Langerhans respond to rising blood glucose levels by secreting insulin that stimulates glucose uptake by peripheral tissues to maintain whole body energy homeostasis. To different extents, failure of β-cell function and/or β-cell loss contribute to the development of Type 1 and Type 2 diabetes. Chronically elevated glycaemia and high circulating free fatty acids, as often seen in obese diabetics, accelerate β-cell failure and the development of the disease. MiRNAs are essential for endocrine development and for mature pancreatic β-cell function and are dysregulated in diabetes. In this review, we summarize the different molecular mechanisms that control miRNA expression and function, including transcription, stability, posttranscriptional modifications, and interaction with RNA binding proteins and other non-coding RNAs. We also discuss which of these mechanisms are responsible for the nutrient-mediated regulation of the activity of β-cell miRNAs and identify some of the more important knowledge gaps in the field.

Pancreatic beta cells form a highly connected network within the islet, with transcriptomic and functional heterogeneity existing between individual cells. A sub-population of leader cells has been proposed to act as functional hubs, controlling islet responses to glucose and other secretagogues. Using high speed confocal Ca2+ imaging, we aimed here to examine (a) the role of these cells in wave propagation and (b) their stability over time. Methods: Islets were isolated from C57BL6 male mice expressing the recombinant Ca2+ probe GCaMP6f selectively in the beta cell (Ins1Cre:GCaMP6f/f). Islets were then infected with an adenovirus expressing photo-activatable- (PA) mCherry. Ca2+ imaging was performed across a single islet plane in modified KREBS-Ringer medium at 11mM glucose using a Zeiss LSM 810 laser-scanning microscope and data acquisition at 5 Hz. Cells were photopainted by excitation of PA-mCherry at 405nm. Results: Leader cells were identified as those exhibiting the first detectable increase in Ca2+ signal during subsequent Ca2+ waves. The great majority of islets (85%, n=26, three independent experiments) displayed repetitive Ca2+ waves (1.10±0.26 min-1), and 82.9±5.0% of waves propagated directly from an identifiable leader cell. There was an average of 2.5±0.4 leader cells per islet with 62.9±0.70% of waves emanating from a “principal” leader cell. Ca2+ imaging experiments performed 16 h after the original session revealed that 37±0.08% (n=4) of waves emanated from the same, now photopainted, leader cell. Conclusions: These data suggest that (a) a complex, non-binary, heterogeneity exists between leader and follower beta cells, (b) leader behaviour is an intrinsic phenotype of a sub-population of cells with a half-life in this role of ∼12 h. Disclosure P. L. Chabosseau: None. A. Martinez-sanchez: None. I. Leclerc: Consultant; Spouse/Partner; Sun Pharmaceutical Industries Ltd. V. Salem: None. G. A. Rutter: Advisory Panel; Self; Sun Pharmaceutical Industries Ltd.

Impaired pancreatic {beta}-cell function and insulin secretion are hallmarks of type 2 diabetes. MicroRNAs are short non-coding RNAs that silence gene expression, vital for the development and function of endocrine cells. MiR-125b is a highly conserved miRNA abundant in {beta}-cells, though its role in these cells remains unclear. Here, we show that miR-125b expression in human islets correlates with body mass index (BMI) of the donors and is regulated by glucose in an AMP-activated protein kinase-dependent manner in both mice and humans. Using and unbiased high-throughput approach, we identify dozens of direct gene targets, including the transporter of lysosomal hydrolases M6pr and the mitochondrial fission regulator Mtfp1. Whereas inactivation of miR-125b in human beta cells led to shorter mitochondria and improved glucose stimulated insulin secretion, mice over-expressing mir-125b selectively in {beta}-cells displayed defective insulin secretion and marked glucose intolerance. Moreover, the {beta}-cells of these transgenic animals showed strongly reduced insulin content and secretion and contained enlarged lysosomal structures. Thus, miR125b provides a glucose-controlled regulator of organelle dynamics that negatively regulates insulin secretion in {beta}-cells. HighlightsO_LIIslet miR125b correlates with BMI and is regulated by glucose via AMP-activated protein kinase in {beta}-cells C_LIO_LImiR125b targets dozens of genes including those involved in the regulation of mitochondrial (i.e. Mtfp1) and lysosomal (i.e. M6pr) morphology/function C_LIO_LIDeletion of miR125b results in shorten mitochondria and improves glucose stimulated insulin secretion (GSIS) C_LIO_LISelective overexpression of miR125b in {beta}-cells causes the appearance of enlarged lysosomes, reduced GSIS and insulin content, loss of {beta}-cell identity and impaired insulin secretion and glucose tolerance in vivo C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/444559v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1772ccdorg.highwire.dtl.DTLVardef@11e6e76org.highwire.dtl.DTLVardef@e62b81org.highwire.dtl.DTLVardef@132405c_HPS_FORMAT_FIGEXP M_FIG C_FIG

Aims/hypothesis Transcription factor 7-like 2 (TCF7L2) is a downstream effector of the Wnt/β-catenin signalling pathway implicated in type 2 diabetes risk through genome-wide association studies. Although its expression is critical for adipocyte development, the potential roles of changes in adipose tissue TCF7L2 levels in diabetes risk are poorly defined. Here, we investigated whether forced changes in Tcf7l2 expression in adipocytes affect whole body glucose or lipid metabolism and crosstalk between disease-relevant tissues. Methods Tcf7l2 was selectively ablated in mature adipocytes in C57BL/6J mice using Cre recombinase under Adipoq promoter control to recombine Tcf7l2 alleles floxed at exon 1 (referred to as aTCF7L2 mice). aTCF7L2 mice were fed normal chow or a high-fat diet for 12 weeks. Glucose and insulin sensitivity, as well as beta cell function, were assessed in vivo and in vitro. Levels of circulating NEFA, selected hormones and adipokines were measured using standard assays. Results Reduced TCF7L2 expression in adipocytes altered glucose tolerance and insulin secretion in male but not in female mice. Thus, on a normal chow diet, male heterozygote knockout mice (aTCF7L2het) exhibited impaired glucose tolerance at 16 weeks (p = 0.03) and increased fat mass (1.4 ± 0.1-fold, p = 0.007) but no changes in insulin secretion. In contrast, male homozygote knockout (aTCF7L2hom) mice displayed normal body weight but impaired oral glucose tolerance at 16 weeks (p = 0.0001). These changes were mechanistically associated with impaired in vitro glucose-stimulated insulin secretion (decreased 0.5 ± 0.1-fold vs control mice, p = 0.02) and decreased levels of the incretins glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide (0.6 ± 0.1-fold and 0.4 ± 0.1-fold vs control mice, p = 0.04 and p p = 0.03 and p = 0.05, respectively). Following exposure to a high-fat diet for 12 weeks, male aTCF7L2hom mice exhibited reduced in vivo glucose-stimulated insulin secretion (0.5 ± 0.1-fold vs control mice, p = 0.02). Conclusions/interpretation Loss of Tcf7l2 gene expression selectively in adipocytes leads to a sexually dimorphic phenotype, with impairments not only in adipocytes, but also in pancreatic islet and enteroendocrine cells in male mice only. Our findings suggest novel roles for adipokines and incretins in the effects of diabetes-associated variants in TCF7L2, and further illuminate the roles of TCF7L2 in glucose homeostasis and diabetes risk.

Long non-coding RNAs (lncRNAs) are emerging as crucial regulators of {beta}-cell development and function. Consequently, the mis-expression of members of this group may contribute to the risk of type 2 diabetes (T2D). Here, we investigate roles for an antisense lncRNA expressed from the Pax6 locus (annotated as Pax6os1 in mice and PAX6-AS1 in humans) in {beta}-cell function. The transcription factor Pax6 is required for the development of pancreatic islets and maintenance of a fully differentiated {beta}-cell phenotype. Pax6os1/PAX6-AS1 expression was increased in pancreatic islets and {beta}-cell lines at high glucose concentrations, in islets from mice fed a high fat diet, and in those from patients with type 2 diabetes. Silencing or deletion of Pax6os1/PAX6-AS1 in MIN6 cells and EndoC-{beta}H1cells, respectively, upregulated {beta}-cell signature genes, including insulin. Moreover, shRNA-mediated silencing of PAX6-AS1 in human islets not only increased insulin mRNA, but also enhanced glucose-stimulated insulin secretion and calcium dynamics. In contrast, inactivation of Pax6os1 in mice was largely without effect on glucose homeostasis, though female Pax6os1 null mice on high fat diet (HFD) showed a tendency towards enhanced glucose clearance. Together, our results suggest that increased expression of PAX6-AS1 at high glucose levels may contribute to {beta}-cell dedifferentiation and failure in some forms of type 2 diabetes. Thus, targeting PAX6-AS1 may provide a promising strategy to enhance insulin secretion and improve glucose homeostasis in type 2 diabetes.

MicroRNAs (miRNAs) play key roles in cartilage development and homeostasis and are dysregulated in osteoarthritis. MiR-145 modulation induces profound changes in the human articular chondrocyte (HAC) phenotype, partially through direct repression of SOX9. Since miRNAs can simultaneously silence multiple targets, we aimed to identify the whole targetome of miR-145 in HACs, critical if miR-145 is to be considered a target for cartilage repair. We performed RIP-seq (RNA-immunoprecipitation and high-throughput sequencing) of miRISC (miRNA-induced silencing complex) in HACs overexpressing miR-145 to identify miR-145 direct targets and used cWords to assess enrichment of miR-145 seed matches in the identified targets. Further validations were performed by RT-qPCR, Western immunoblot, and luciferase assays. MiR-145 affects the expression of over 350 genes and directly targets more than 50 mRNAs through the 3&prime, UTR or, more commonly, the coding region. MiR-145 targets DUSP6, involved in cartilage organization and development, at the translational level. DUSP6 depletion leads to MMP13 upregulation, suggesting a contribution towards the effect of miR-145 on MMP13 expression. In conclusion, miR-145 directly targets several genes involved in the expression of the extracellular matrix and inflammation in primary chondrocytes. Thus, we propose miR-145 as an important regulator of chondrocyte function and a new target for cartilage repair.

Aims/hypothesis Mitochondrial oxidative metabolism is central to glucose-stimulated insulin secretion (GSIS). Whether Ca2+ uptake into pancreatic beta cell mitochondria potentiates or antagonises this process is still a matter of debate. Although the mitochondrial Ca2+ importer (MCU) complex is thought to represent the main route for Ca2+ transport across the inner mitochondrial membrane, its role in beta cells has not previously been examined in vivo. Methods Here, we inactivated the pore-forming subunit of the MCU, encoded by Mcu, selectively in mouse beta cells using Ins1Cre-mediated recombination. Whole or dissociated pancreatic islets were isolated and used for live beta cell fluorescence imaging of cytosolic or mitochondrial Ca2+ concentration and ATP production in response to increasing glucose concentrations. Electrophysiological recordings were also performed on whole islets. Serum and blood samples were collected to examine oral and i.p. glucose tolerance. Results Glucose-stimulated mitochondrial Ca2+ accumulation (pppMcu-null (βMcu-KO) animals, in vitro, as compared with wild-type (WT) mice. Interestingly, cytosolic Ca2+ concentrations increased (pMcu-KO animals. βMcu-KO mice displayed impaired in vivo insulin secretion at 5 min (ppMcu-KO ( Conclusions/interpretation MCU is crucial for mitochondrial Ca2+ uptake in pancreatic beta cells and is required for normal GSIS. The apparent compensatory mechanisms that maintain glucose tolerance in βMcu-KO mice remain to be established.

ObjectiveMicroRNAs play a key role in biological processes, including cartilage development and homeostasis and are dysregulated in many diseases, including osteoarthritis. MiR-145 modulation induces profound changes in the human articular chondrocyte (HAC) phenotype, partially through direct repression of SOX9. Since miRNAs can simultaneously silence multiple targets, we aimed to identify the whole targetome of miR-145 in HACs. This information is critical if miR-145 is to be considered a target for cartilage repair.MethodsRIP-seq (RNA-immunoprecipitation plus HT-sequencing) of miRISC (miRNA-induced silencing complex) was performed in HACs overexpressing miR-145 to identify miR-145 direct targets. The motif discovery method cWords was used to assess enrichment on miR-145 seed matches in the identified targets. RT-qPCR, Western (immuno-)blot and luciferase assays were used to validate miRNA-target interactions.ResultsMiR-145 overexpression affects the expression of over 350 genes and directly targets more than 50 mRNAs through the 3’UTR or, more commonly, the coding region.We also demonstrate that miR-145 targets DUSP6, involved in cartilage organization and development, at the translational level. DUSP6 depletion using specific siRNAs lead to MMP13 up-regulation, suggesting that miR-145-mediated DUSP6 depletion contributes to the effect of miR-145 on MMP13 expression.ConclusionWe demonstrate that miR-145 directly targets several genes in primary chondrocytes including those involved in the expression of the extracellular matrix and inflammation. Thus, we propose miR-145 as an important regulator of chondrocyte function and a new target for cartilage repair.

Global rates of diabetes mellitus are increasing, and treatment of the disease consumes a growing proportion of healthcare spending across the world. Pancreatic β-cells, responsible for insulin production, decline in mass in Type 1 and, to a more limited degree, in Type 2 diabetes. However, the extent and rate of loss in both diseases differs between patients resulting in the need for the development of novel diagnostic tools, which could quantitatively assess changes in mass of β-cells over time and potentially lead to earlier diagnosis and improved treatments. Exendin-4, a potent analogue of glucagon-like-peptide 1 (GLP-1), binds to the receptor GLP-1R, whose expression is enriched in β-cells. GLP-1R has thus been used in the past as a means of targeting probes for a wide variety of imaging modalities to the endocrine pancreas. However, exendin-4 conjugates designed specifically for MRI contrast agents are an under-explored area. In the present work, the synthesis and characterization of an exendin-4-dota(ga)-Gd(III) complex, GdEx, is reported, along with its in vivo behaviour in healthy and in β-cell-depleted C57BL/6J mice. Compared to the ubiquitous probe, [Gd(dota)](-), GdEx shows selective uptake by the pancreas with a marked decrease in accumulation observed after the loss of β-cells elicited by deleting the microRNA processing enzyme, DICER. These results open up pathways towards the development of other targeted MRI contrast agents based on similar chemistry methodology.

Aims/hypothesis Mitochondrial oxidative metabolism is central to glucose-stimulated insulin secretion (GSIS). Whether Ca2+ uptake into pancreatic beta cell mitochondria potentiates or antagonises this process is still a matter of debate. Although the mitochondrial Ca2+ importer (MCU) complex is thought to represent the main route for Ca2+ transport across the inner mitochondrial membrane, its role in beta cells has not previously been examined in vivo. Methods Here, we inactivated the pore-forming subunit of the MCU, encoded by Mcu, selectively in mouse beta cells using Ins1Cre-mediated recombination. Whole or dissociated pancreatic islets were isolated and used for live beta cell fluorescence imaging of cytosolic or mitochondrial Ca2+ concentration and ATP production in response to increasing glucose concentrations. Electrophysiological recordings were also performed on whole islets. Serum and blood samples were collected to examine oral and i.p. glucose tolerance. Results Glucose-stimulated mitochondrial Ca2+ accumulation (p

Mitochondrial oxidative metabolism is central to glucose-stimulated insulin secretion (GSIS). Whether Ca2+uptake into pancreatic β-cell mitochondria potentiates or antagonises this process is still a matter of debate. Although the mitochondrial importer (MCU) complex is thought to represent the main route for Ca2+transport across the inner mitochondrial membrane, its role in β-cells has not previously been examinedin vivo. Here, we inactivated the pore-forming subunit MCUa (MCU) selectively in the β-cell in mice usingIns1Cre-mediated recombination. Glucose-stimulated mitochondrial Ca2+accumulation, ATP production and insulin secretion were strongly (pMCUnull animals (βMCU-KO)in vitro. Interestingly, cytosolic Ca2+concentrations increased (pin vivoinsulin secretion at 5 (p2+uptake in pancreatic β-cells and is required for normal GSIS. The apparent compensatory mechanisms which maintain glucose tolerance in βMCU-KO mice remain to be established.

AMP-activated protein kinase (AMPK) is an important cellular energy sensor in regulation of metabolic activity. AMPK activation improves glucose homeostasis by increasing muscle glucose uptake and reducing hepatic glucose production. Although the effects on glucose-stimulated insulin secretion in the pancreatic β cells are still controversial. Our aim was to investigate the regulation of insulin secretion in pancreatic β cells using specific pharmacological AMPK activators and in mouse models of constitutive activation. We used a chronic global AMPK (γ1D316A) and a pancreatic β cell specific (β-D316A) activation mouse model that express the AMPK γamma1 isoform with a D316A mutation under the control of β-actin and ins1 promoter respectively. We tested how chronic AMPK activation impacted insulin secretion on C57BL6 mouse isolated islets using direct activators PF-06409577 (β1 isoform-selective) and RA089 (binds both β1 and β2 isoforms). Global activation of AMPK reduced insulin secretion from islets isolated from γ1D316A mice on chow and high-fat diet (2.4-fold, p Disclosure M. Nguyen-Tu: None. J.P. Harris: None. A. Martinez-Sanchez: None. I. Leclerc: None. G.A. Rutter: Consultant; Self; Sun Pharma.

miRNAs are tiny RNAs that repress gene expression post-transcriptionally, necessary for normal endocrine function. AMPK is essential for glucose homeostasis and a suggested target of metformin. β-cell-specific inactivation of AMPK in mice (AMPKdKO) impairs glucose homeostasis and β-cell function. AMPKdKO mice present dysregulation of several miRNAs that may contribute to these defects, including miR-125b (∼2.5 fold upregulated). High levels of miR-125b are associated with hyperglycaemia (HbA1c) in prediabetic, T2D and T1D subjects, suggesting miR-125b as a biomarker or contributor to the development of diabetes. The role of miR-125b in β-cells remains elusive although in other cell types regulates apoptosis, growth and differentiation. We aim to determine miR-125b function in β-cells and the role of glucose and AMPK in the regulation of its expression. miR-125b level in human islets strongly correlates with BMI and is induced by glucose in both mouse and human islets, while feeding mice a ketogenic (low sugar) diet reduced islet miR-125b. These effects disappeared in islets with β-cell specific deletion of AMPK whereas treatment of human islets with highly specific AMPK activators significantly decreased miR-125b, indicating that glucose regulates miR-125b via AMPK in both mice and humans. miR-125b overexpression in MIN6 cells limited cytokine-induced apoptosis whilst strongly reducing insulin content. RNA-Seq on these cells suggests a role for miR-125b in metabolism, respiration and cytokine receptor interactions and RNA-IP of the miRNA-induced silencing complex revealed novel miR-125b targets such as mannose-6-phosphate receptor (MPR), important for enzyme sorting within secretory granules. Preliminary data show that β-cell-specific inhibition of miR-125b using a molecular “sponge” improves glucose tolerance in mice fed a high-fat diet. In summary, we reveal miR-125b as an important regulator of β-cell function that might contribute to the deleterious effects of hyperglycaemia on β-cells. Disclosure R. Cheung: None. G. Pizza: None. D.M. Rolando: None. P.L. Chabosseau: None. M. Nguyen-Tu: None. I. Leclerc: None. G.A. Rutter: Consultant; Self; Sun Pharma. A. Martinez-Sanchez: None. Funding Medical Research Council UK

Objective To investigate the function of microRNA-138 (miR-138) in human articular chondrocytes (HACs). Methods The expression of miR-138 in intact cartilage and cultured chondrocytes and the effects of miR-138 overexpression on chondrocyte marker genes were investigated. Targets of miR-138 relevant to chondrocytes were identified and verified by overexpression of synthetic miRNA mimics and inhibitors, luciferase assays, chromatin immunoprecipitation, and RNA immunoprecipitation of native argonaute 2, using quantitative polymerase chain reaction, Western blotting, and luciferase assays. Results Expression levels of miR-138 were maintained at relatively low levels in intact human cartilage but were greatly increased upon loss of the differentiated phenotype in culture, with a concomitant decrease in the major cartilage extracellular matrix component COL2A1. We showed that miR-138 is able to repress the expression of COL2A1 by directly targeting Sp-1 and hypoxia-inducible factor 2α (HIF-2α), 2 transcription factors that are essential for COL2A1 transcription. We further demonstrated a direct association of these targets with miR-138 in the RNA-induced silencing complex and confirmed binding of Sp-1 to the COL2A1 promoter region in HACs. Conclusion We propose that an evolutionary pressure helps to suppress expression levels of miR-138 in human cartilage, thus enabling expression of appropriate tissue-specific matrix genes. Inhibition of miR-138 may serve as a potential therapeutic strategy to maintain the chondrocyte phenotype or reduce the progression of dedifferentiation in cultured HACs.

Pancreatic β-cells are the body's sole source of circulating insulin and essential for the maintenance of blood glucose homeostasis. Levels of up to 66 “disallowed” genes, which are strongly expressed and play housekeeping roles in most other mammalian tissues, are unusually low in β-cells. The molecular mechanisms involved in repressing these genes are largely unknown. Here, we explore the role in gene disallowance of microRNAs (miRNAs), a type of small noncoding RNAs that silence gene expression at the posttranscriptional level and are essential for β-cell development and function. To selectively deplete miRNAs from adult β-cells, the miRNA-processing enzyme DICER was inactivated by deletion of the RNase III domain with a tamoxifen-inducible Pdx1CreER transgene. In this model, β-cell dysfunction was apparent 2 weeks after recombination and preceded a decrease in insulin content and loss of β-cell mass. Of the 14 disallowed genes studied, quantitative RT-quantitative real-time PCR revealed that 6 genes (Fcgrt, Igfbp4, Maf, Oat, Pdgfra, and Slc16a1) were up-regulated (1.4- to 2.1-fold, P < .05) at this early stage. Expression of luciferase constructs bearing the 3′-untranslated regions of the corresponding mRNAs in wild-type or DICER-null β-cells demonstrated that Fcgrt, Oat, and Pdgfra are miRNA direct targets. We thus reveal a role for miRNAs in the regulation of disallowed genes in β-cells and provide evidence for a novel means through which noncoding RNAs control the functional identity of these cells independently of actions on β-cell mass.

MicroRNAs (miRNAs) are small non-coding RNA molecules of 21-23 nucleotides that control gene expression at the post-transcriptional level. They have been shown to play a vital role in a wide variety of biological processes and dysregulated expression of miRNAs is observed in many pathologies. Understanding the mechanism of action and identifying functionally important mRNA targets of a specific miRNA are essential to unravelling its biological function and to assist miRNA-based drug development. This review summarizes the current understanding of the mechanistic aspects of miRNA-mediated gene repression and focuses on the different approaches for miRNA target identification that have been proposed in recent years.

Encoding acyl-CoA thioesterase-7 (Acot7) is one of ∼60 genes expressed ubiquitously across tissues but relatively silenced, or disallowed, in pancreatic β-cells. The capacity of ACOT7 to hydrolyze long-chain acyl-CoA esters suggests potential roles in β-oxidation, lipid biosynthesis, signal transduction, or insulin exocytosis. We explored the physiological relevance of β-cell–specific Acot7 silencing by re-expressing ACOT7 in these cells. ACOT7 overexpression in clonal MIN6 and INS1(832/13) β-cells impaired insulin secretion in response to glucose plus fatty acids. Furthermore, in a panel of transgenic mouse lines, we demonstrate that overexpression of mitochondrial ACOT7 selectively in the adult β-cell reduces glucose tolerance dose dependently and impairs glucose-stimulated insulin secretion. By contrast, depolarization-induced secretion was unaffected, arguing against a direct action on the exocytotic machinery. Acyl-CoA levels, ATP/ADP increases, membrane depolarization, and Ca2+ fluxes were all markedly reduced in transgenic mouse islets, whereas glucose-induced oxygen consumption was unchanged. Although glucose-induced increases in ATP/ADP ratio were similarly lowered after ACOT7 overexpression in INS1(832/13) cells, changes in mitochondrial membrane potential were unaffected, consistent with an action of Acot7 to increase cellular ATP consumption. Because Acot7 mRNA levels are increased in human islets in type 2 diabetes, inhibition of the enzyme might provide a novel therapeutic strategy.

p27(kip1) (p27) is a cell cycle inhibitor and tumor suppressor whose expression is tightly regulated in the cell. Translational control of p27 mRNA has emerged as a prominent mechanism to regulate p27 expression during differentiation, quiescence, and cancer progression. The microRNAs miR-221 and miR-222 repress p27 expression in various cancer cells, and this repression promotes tumor cell proliferation. In addition, the presence of an internal ribosome entry site in the 5' untranslated region (UTR) of p27 mRNA has been reported. Here, we show that p27 mRNA is translated via a cap-dependent mechanism in HeLa and HL60 cells and that the previously reported IRES activity can be attributed to cryptic promoters in the sequence corresponding to the p27 5' UTR. Furthermore, cap-dependent translation of p27 mRNA is repressed by miR-181a in undifferentiated HL60 cells. Repression by miR-181a is relieved during differentiation of HL60 into monocyte-like cells, allowing the accumulation of p27, which is necessary to fully block cell cycle progression and reach terminal differentiation. These results identify miR-181a as a regulator of p27 mRNA translation during myeloid cell differentiation.

Fully differentiated pancreatic β cells are essential for normal glucose homeostasis in mammals. Dedifferentiation of these cells has been suggested to occur in type 2 diabetes, impairing insulin production. Since chronic fuel excess ("glucotoxicity") is implicated in this process, we sought here to identify the potential roles in β-cell identity of the tumor suppressor liver kinase B1 (LKB1/STK11) and the downstream fuel-sensitive kinase, AMP-activated protein kinase (AMPK). Highly β-cell-restricted deletion of each kinase in mice, using an Ins1-controlled Cre, was therefore followed by physiological, morphometric, and massive parallel sequencing analysis. Loss of LKB1 strikingly (2.0-12-fold, E

microRNAs are a large and essential class of gene regulators that play key roles in development, homeostasis, and disease. They are necessary for normal skeletal development, and their expression is altered in arthritis. However, the specific role of individual microRNAs is only beginning to be unraveled. Using microRNA expression profiling in healthy human articular cartilage cells (chondrocytes), we identified miR-1247 expression as highly correlated with that of the differentiated cell phenotype. Transcribed from the DLK1-DIO3 locus, the function of miR-1247 is completely unknown. In mice its expression level was relatively high in cartilage tissue, and correlated with cartilage-associated microRNA miR-675 across a range of 15 different mouse tissues. To further probe miR-1247 function, overexpression and inhibition studies were performed in isolated human chondrocytes. Modulation of miR-1247 was found to exert profound phenotypic effects altering expression levels of cartilage master regulator transcription factor SOX9. SOX9 is essential for cartilage development and subsequent function throughout life, and mutations in this gene result in severe dwarfism. Putative miR-1247 binding sites were further investigated using luciferase reporter assays, which indicated binding of miR-1247 to a highly conserved region in the coding sequence of SOX9 but not in its 3′-UTR. Interestingly, depletion of SOX9 in human chondrocytes resulted in increased levels of the mature, processed microRNA, suggesting a negative feedback loop between miR-1247 and its target SOX9. Background: The function of miR-1247 was heretofore unknown. Results: We show that miR-1247 directly targets SOX9, a transcription factor essential for cartilage formation and function. Conclusion: miR-1247 may be an important regulator of cartilage function. Significance: miR-1247 is a potential new target for joint repair.

Articular cartilage enables weight bearing and near friction-free movement in the joints. Critical to its function is the production of a specialized, mechanocompetent extracellular matrix controlled by master regulator transcription factor SOX9. Mutations in SOX9 cause campomelic dysplasia, a haploinsufficiency disorder resulting in severe skeletal defects and dwarfism. Although much is understood about how SOX9 regulates cartilage matrix synthesis and hence joint function, how this master regulator is itself regulated remains largely unknown. Here we identify a specific microRNA, miR-145, as a direct regulator of SOX9 in normal healthy human articular chondrocytes. We show that miR-145 directly represses SOX9 expression in human cells through a unique binding site in its 3'-UTR not conserved in mice. Modulation of miR-145 induced profound changes in the human chondrocyte phenotype. Specifically, increased miR-145 levels cause greatly reduced expression of critical cartilage extracellular matrix genes (COL2A1 and aggrecan) and tissue-specific microRNAs (miR-675 and miR-140) and increased levels of the hypertrophic markers RUNX2 and MMP13, characteristic of changes occurring in osteoarthritis. We propose miR-145 as an important regulator of human chondrocyte function and a new target for cartilage repair.

miRNAs have been shown to be essential for normal cartilage development in the mouse. However, the role of specific miRNAs in cartilage function is unknown. Using rarely available healthy human chondrocytes (obtained from 8 to 50 year old patients), we detected a most highly abundant primary miRNA H19, whose expression was heavily dependent on cartilage master regulator SOX9. Across a range of murine tissues, expression of both H19- and H19-derived miR-675 mirrored that of cartilage-specific SOX9. miR-675 was shown to up-regulate the essential cartilage matrix component COL2A1, and overexpression of miR-675 rescued COL2A1 levels in H19- or SOX9-depleted cells. We thus provide evidence that SOX9 positively regulates COL2A1 in human articular chondrocytes via a previously unreported miR-675-dependent mechanism. This represents a novel pathway regulating cartilage matrix production and identifies miR-675 as a promising new target for cartilage repair.