Role of microRNAs in the age-associated decline of pancreatic beta cell function in rat islets
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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.
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.
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.
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.
KeywordsAgeing Apoptosis Beta cell Diabetes Insulin secretion MicroRNA Pancreatic islet Proliferation
Median number of miRNA recognition elements
Platelet-derived growth factor
Platelet-derived growth factor receptor α
Quantitative real-time PCR
Insulin secretion from pancreatic beta cells plays a central role in blood glucose homeostasis and metabolism control. Reduced sensitivity of insulin target tissues and a consequent rise in insulin demand is normally compensated by expansion of beta cells and an increase in their secretory activities. Failure in this compensatory process results in the release of insufficient insulin to cover the organism’s needs and the development of type 2 diabetes . Ageing is a risk factor for several metabolic diseases, including type 2 diabetes. Indeed, ageing can affect both insulin secretion and insulin action, and predisposes to glucose intolerance and diabetes . Moreover, ageing is associated with impaired proliferation and increased sensitivity of beta cells to apoptosis , reducing their capacity to cope with an insulin-resistance state. At present, the mechanisms underlying these phenomena are not fully understood, but changes in the expression of genes coding for key proteins have been reported to be involved in the age-associated decline of beta cell function.
MicroRNAs (miRNAs) are short non-coding RNAs that bind to the 3′ untranslated region (UTR) of target mRNAs, causing translational repression and/or messenger degradation . During the past decade, several studies have demonstrated the involvement of miRNAs in the regulation of beta cell function and survival [5, 6, 7]. Although the role of miRNAs in the regulation of beta cell activities has been investigated in various conditions, including pregnancy and obesity [8, 9, 10, 11], the potential contribution of these molecules to age-associated beta cell impairment has not, as yet, been explored.
In this study, we compared the islet miRNA profiles of 3- and 12-month-old rats. Several differentially expressed miRNAs were identified and their roles in beta cell secretion, proliferation and survival upon chronic exposure to proapoptotic conditions were systematically investigated.
Prolactin, IL-1β, exendin-4 and platelet-derived growth factor (PDGF)-AA were obtained from Sigma-Aldrich (St Louis, MO, USA). TNF-α was obtained from Enzo Life Sciences (Lausanne, Switzerland) and IFNγ was obtained from R&D Systems (Minneapolis, MN, USA).
Wistar rats were from Janvier Laboratories (Le Genest St Ile, France). All procedures were approved by the Swiss Veterinary Office and were in accordance with National Institutes of Health guidelines.
Islet isolation and cell culture
Islets were isolated by collagenase digestion  and cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) . Dissociated islet cells were obtained by trypsin digestion . Human islets were received from the Cell Isolation and Transplantation Center (University of Geneva, Geneva, Switzerland). Dissociated human islet cells were cultured in CMRL medium (Invitrogen) . The rat insulin-secreting cell line INS832/13 was cultured as previously described .
Profiling of miRNA and mRNA
RNA was isolated using the miRNeasy Kit (Qiagen, Hombrechtikon, Switzerland). MiRNA expression profiling was carried out using Agilent Technologies (Santa Clara, CA, USA) miRNA Microarrays . Profiling of mRNAs was carried out by Arraystar (Rockville, MD, USA).
Measurement of miRNA and mRNA expression
MiRNA expression was assessed using the miRCURY LNA Universal RT miRNA PCR kit (Exiqon, Vedbaek, Denmark). Measurements of mRNA levels were performed by quantitative real-time PCR (qPCR; Bio-Rad, Reinach, Switzerland) with custom-designed primers (Microsynth, Balgach, Switzerland) (see Electronic Supplementary Material [ESM] Methods). MiRNA expression was normalised to the level of U6 or miR-7, while mRNA expression was normalised to 18S. The level of selected miRNAs in cadaveric human islets from 11 normoglycaemic donors, and their association with age, was assessed in our previously published global miRNA profiling dataset .
Transfection and modulation of miRNA levels
INS832/13 cells and primary rat or human dissociated islet cells were transfected with Lipofectamine 2000 (Invitrogen), either with RNA oligonucleotide duplexes (Eurogentec, Seraing, Belgium) corresponding to the sequence of the miRNAs (overexpression) or with single-stranded miScript miRNA Inhibitors (Qiagen) that specifically inhibit endogenous miRNAs . A custom-designed small interfering RNA duplex directed against green fluorescent protein (Eurogentec) and a miScript reference miRNA (Qiagen) were used as respective controls.
The insulin content of and insulin secretion by dissociated rat islet cells were measured by ELISA .
Cell death assessment
Rat and human islet cells were incubated with 1 μg/ml Hoechst 33342 (Invitrogen) for 1 min. At least 103 cells/condition were analysed under fluorescence microscopy (AxioCam MRc 5, Zeiss, Feldbach, Switzerland) to score the fraction of cells displaying pycnotic nuclei.
Cells were cultured on poly-l-lysine-coated glass coverslips. They were fixed with cold methanol, permeabilised with 0.5% saponin (Sigma-Aldrich) and exposed for 1 h to antibodies against Ki67 (1:400) (Abcam, Cambridge, UK) and insulin (1:10,000) (Millipore, Zug, Switzerland). Coverslips were then incubated for 1 h with anti-rabbit Alexa-Fluor-488 and anti-mouse Alexa-Fluor-555 antibodies (Invitrogen). Images were collected on an AxioVision fluorescence microscope.
A luciferase reporter construct was generated by inserting 212 nucleotides of the 3′ UTR sequence of rat Pdgfra surrounding the putative binding site of miR-34a between the XhoI and EcoRI sites of psiCHECK-1 (ESM Methods). Luciferase activity was measured using a dual-luciferase reporter assay (Promega, Madison, WI, USA). Firefly luciferase activity was normalised for transfection efficiency with the SV40-driven Renilla activity generated by pGL3-Basic (Promega).
Cells were lysed in Laemmli buffer. Lysates were resolved by SDS-PAGE, transferred to a PVDF membrane and detected using antibodies against PDGF receptor α (PDGFRα) (catalogue no. 3174; Cell Signaling Technology, Danvers, MA, USA) and α-tubulin (Fluka Chemie, Buchs, Switzerland) . After 1 h incubation at room temperature with horseradish peroxidase-conjugated secondary antibodies (Fluka Chemie), membranes were revealed by chemiluminescence (Immobilon, Millipore) using the ChemiDoc XRS+ System (Bio-Rad Laboratories).
miRNA target enrichment analysis
For each differentially expressed miRNA, we estimated the median number of miRNA recognition elements (Mobs) predicted, using TargetScan (version 6.2) , in the 3′ UTR (rn6, downloaded from UCSC  on 10 April 2015) of n up- or downregulated genes. To obtain an empirical p value associated with each Mobs, we independently estimated 1,000 times the median TargetScan predicted density of miRNA recognition elements for N regions of matching length randomly sampled from the 3′ UTRs of rat islet mRNAs that were not differentially expressed.
Statistical differences were tested using Student’s t test or for multiple comparisons, with ANOVA followed by a post hoc Dunnett test, with a discriminating p value of 0.05 (SAS statistical package, Cary, NC, USA).
To determine whether these phenotypic traits were linked to transcriptomic differences, we used microarray analysis to compare the global mRNA profiles of the islets isolated from young and old rats. We detected 632 upregulated and 397 downregulated genes (fold change >2.0; nominal p < 0.05, n = 3) in the islets of 12-month-old rats (ESM Tables 1, 2; microarray data are deposited in Gene Expression Omnibus under the accession code GSE72466). These changes are unlikely to reflect differences in islet composition, since the islet β-cell content is not modified in 12-month-old animals . Among the upregulated mRNAs, pathway analysis revealed an enrichment of genes involved in insulin secretion, mitogen-activated protein kinase signalling and MODY (ESM Table 3). Changes in the expression of selected genes belonging to these pathways were verified by qPCR (ESM Fig. 2). In particular, we confirmed the induction of genes involved in insulin secretion (such as the calcium-channel subunits Cacna1c and Cacna1d) and MODY (Slc2a2 and Neurod1), and encoding the transcription factor p53 (Tp53).
We then verified whether the observed modifications in miRNA expression are specific for islet cells. Most of the selected miRNAs displayed expression profiles in insulin target tissues (liver, skeletal muscle and adipose tissue) and brain that differed from those of islets (ESM Table 5). In contrast, and in line with the literature [26, 27], miR-34a was upregulated in the liver and brain of older rats relative to 3-month-old rats (Fig. 3b, c).
Ageing is a risk factor for the development of type 2 diabetes. In fact, it is associated with a progressive decrease in insulin sensitivity and a decline in beta cell function . The mechanisms underlying these effects are not fully understood, but are likely to involve changes in gene expression. Here, we investigated the transcriptomic modifications occurring in islets of 12-month-old rats. In agreement with other studies , we did not observe alterations in insulin secretion or apoptosis in the islets of these animals, suggesting that beta cells preserve the capacity to accomplish their basic tasks. However, beta cells from 12-month-old rats were unable to respond to mitogens. The loss of beta cell proliferation is probably not a significant handicap under normal conditions, but might constitute a major obstacle to the achievement of blood glucose homeostasis under conditions of insulin resistance requiring compensatory beta cell mass expansion.
Our data indicate that the expression of several protein-coding genes and miRNAs is already modified in the islets of 12-month-old rats. Some of these transcriptomic changes are likely to have a positive impact on beta cell function. For instance, we detected the upregulation of genes involved in insulin release, such as those coding for calcium-channel subunits [35, 36, 37] and the glucose transporter GLUT-2 (Slc2a2) . These data suggest the existence of compensatory mechanisms operating to maintain efficient insulin secretion in 12-month-old animals.
In addition to differences in protein-coding genes, the islets of 12-month-old rats also displayed modifications in the miRNA profile. When changes in the levels of selected miRNAs were reproduced in islet cells of young animals, none of them affected insulin biosynthesis or secretion. In cell lines, overexpression of miR-124a and miR-34a has previously been reported to inhibit insulin secretion [25, 28, 39] but this observation was not confirmed in another study, although glucose-induced Ca2+ fluxes were affected by miR-124a overexpression . These discrepant findings might be due to differences in the level of overexpression achieved after cell transfection. Moreover, because of transcriptomic differences, these miRNAs might have distinct impacts on insulin secretion in primary beta cells and tumoral beta cell lines. These miRNAs reduce the expression of key components of the machinery of insulin exocytosis [25, 28, 39]. Thus, although overexpression in beta cells of young animals did not significantly impair insulin release, we cannot exclude the possibility that the presence of higher levels of miR-34a or miR-124a would exacerbate the secretory decline of ageing beta cells.
Several of the miRNAs displaying expression changes in ageing animals affect the survival of beta cells. Indeed, downregulation of miR-130b or induction of miR-383 improved the survival of beta cells under proapoptotic conditions. Similar protective effects have already been reported for other miRNAs under insulin-resistance conditions [8, 9]. The changes in the levels of these miRNAs might be sufficient to compensate for the proapoptotic effect elicited by the concomitant upregulation of miR-34a. This suggests that a balance between the action of proapoptotic and antiapoptotic miRNAs prevents a net loss of beta cells in 12-month-old rats.
The most striking defect observed in beta cells of 12-month-old rats was the loss of proliferation in response to mitotic stimuli. Interestingly, overexpression of miR-34a or blockade of miR-181a was sufficient to reproduce this phenotypic trait in the islets of younger animals. The mechanism through which the downregulation of miR-181a contributes to the age-associated impairment of beta cell proliferation remains to be established. However, analysis of differentially expressed genes in the islets of 12-month-old rats revealed an enrichment of miR-181a targets in the upregulated mRNAs and a reduction in those that were downregulated. Thus, attenuation of the repressive activity of this miRNA appears to contribute to transcriptomic modifications occurring in the islets of ageing animals. The islet gene-expression profile of ageing animals also appears to be influenced by the induction of miR-34a. Indeed, the 3′ UTR of the upregulated genes was depleted from potential recognition sequences for this miRNA, and the putative miR-34a targets tended to be more frequent in genes downregulated in ageing. Some of the targets of miR-34a might be directly involved in the proliferative defect. Indeed, we demonstrated that the mRNA coding for PDGFRα is directly targeted by miR-34a. PDGFR signalling plays a critical role in postnatal beta cell mass expansion and in beta cell regeneration . Thus, the translational repression of Pdgfra exerted by the induction of miR-34a is likely to contribute to the loss of proliferative capacity observed during ageing.
The increase in the level of miR-34a in the islets of ageing rats is probably linked to the activation of p53 signalling. Indeed, we have previously reported that, in insulin-secreting cells, this transcription factor binds to the promoter of miR-34a and triggers expression of the miRNA . The p53 pathway is induced by a variety of stresses and plays a pivotal role in cellular senescence and metabolic homeostasis [41, 42]. p53 and miR-34a are linked in a positive-feedback loop to sirtuin-1 (SIRT1). Indeed, p53 induces the expression of miR-34a and the miRNA targets and represses SIRT1, preventing SIRT1-mediated deacetylation of p53 and, in turn, promoting the activity of the transcription factor . Consistent with this model, we found that increases in p53 and miR-34a in the islets of ageing animals were indeed associated with reduced levels of SIRT1.
In addition to being upregulated in islets, the level of miR-34a was also elevated in the liver and brain of old rats, pointing to a general role for this miRNA in the ageing process. Moreover, the expression of this miRNA has been found to be abnormally elevated in islets and insulin target tissues of obese animals [40, 44, 45], contributing to beta cell failure and insulin resistance. Being at the crossroad between ageing and metabolic imbalance, these findings point to miR-34a induction as an important risk factor for the development of type 2 diabetes. This will particularly hold true for individuals expressing single nucleotide polymorphisms within the precursor of miR-34a that result in increased expression of this miRNA .
In this study, we identified a group of islet miRNAs that display expression changes during ageing and are likely to contribute to the progressive failure of beta cells to compensate for insulin resistance. A better understanding of the role and the mode of action of these miRNAs in beta cells might open the way to the development of new strategies for the prevention and/or treatment of type 2 diabetes.
This work was supported by the Swiss National Science Foundation (grant number 310030-146138) (RR) and by the ‘Fondation Francophone pour la Recherche sur le Diabète’ (RR). GAR was supported by a Wellcome Trust Senior Investigator Award (WT098424AIA), an MRC Programme Grant (MR/J0003042/1) and a Royal Society Wolfson Research Merit Award. LWH and JML were supported by a Wellcome Trust project grant (number 089845/Z/09/Z).
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
KT generated the data and wrote the manuscript. CG and FA contributed to the acquisition of data and critically reviewed the manuscript. LWH, JML, ACM and GAR contributed to data acquisition and interpretation, and reviewed the manuscript. RR conceived the experiments, interpreted the data and wrote the manuscript. All authors approved the final version. RR is the guarantor of this work.
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