Pancreatic beta cells are responsible for insulin release which is essential for normal blood glucose homeostasis. Thanks to an unusual metabolic configuration, the β-cells act as nutrient sensors by coupling their glucose metabolism with the insulin secretion, delivering an appropriate quantity in the systemic circulation. When metabolized in the β-cell, glucose promotes the increase of intracellular ATP. This causes the closure of ATP-sensitive K+ channels (KATP) which in turn leads to the depolarization of the plasma membrane causing the opening of voltage-gated Ca2+ channels. The resultant increase in intracellular free Ca2++ both triggers the exocytotic release of the insulin from secretory granules and reinforces the response by stimulating the ATP production by the mitochondria. Other KATP independent mechanisms still incompletely understood, including the inhibition of AMP-activated protein kinase, potentiate this process1,2.
While type I diabetes is a relatively well-characterized disease caused by the autoimmune-mediated destruction of the β-cells, type II diabetes is a less defined condition, where defects in glucose sensing and a loss of β-cells combine to reduce insulin secretion, resulting in aberrant blood glucose levels. A full understanding of the mechanisms underlying insulin secretion is essential to the development of new therapies.
The specific function of the β-cells requires the activation of several genes which are expressed in none or few other tissues. For example, the low affinity hexokinase, glucokinase is preferentially expressed in liver and β-cells. This enzyme facilitates the conversion of glucose to glucose-6-phospate and operates as a physiological glucose sensor thanks to its unique kinetic properties, different to the hexokinases present in other tissues3. More examples include other genes involved in glucose sensing or specific transcription factors such as PDX1 or MafA4. The importance of some of those genes has been highlighted by the occurrence of their mutation in a monogenic type of diabetes known as maturity-onset diabetes of the young (MODY)5 or by the detection of abnormalities in insulin secretion in mice bearing reduced activity of these genes6.
Conversely, it has recently become apparent that some genes serving a "housekeeping" role in most cells and therefore expressed in more or less abundance in most of the other tissues, are absent -or disallowed- in β-cells7. The two founder members of these "disallowed" genes are monocarboxylate transporter-1 (MCT-1) and lactate dehydrogenase A (LDHA) whose expression is very low in β-cells while ubiquitously expressed across other mammalian tissues8,9. All living cells need a continuous production of ATP, normally generated via oxidative phosphorylation-which is oxygen-dependant-in the mitochondria inner membrane. The survival of the cells under anaerobic conditions can occur thanks to anaerobic glycolysis, which involves the reduction of pyruvate to lactate thanks to LDHA and the transport of pyruvate and lactate across the plasma membrane, which is achieved thanks to a monocarboxylate transporter. Accordingly and as an exception, glycolysis is exclusively aerobic in β-cells10 where these two gene products are absent. Posterior studies demonstrated that the low expression of those genes has a double function: First, assuring that pyruvate derived from glycolysis is preferentially directed toward mitochondrial oxidation-reinforcing the ability of glucose to stimulate insulin secretion and, second, to avoid the stimulation of insulin secretion by the pyruvate generated by muscles during physical exercise11. Indeed, mutations within the SLC16A1 (MCT-1) promoter increasing the expression of MCT1 were found in two families suffering of exercise-induced hyperinsulinism (EIHI)12. In this autosomal dominant disorder, vigorous physical exercise causes inappropriate insulin release, leading to hypoglucemia13. Pullen and co-workers generated a transgenic mouse overexpressing Mct-1 and showed that, in response to exercise, transgene induction is enough to prevent the normal inhibition of insulin secretion, mimicking the key features of EIHI and exposing the importance of the absence of this transporter for the normal control of insulin secretion11.
The laboratory here suggested as a host and others have recently described more than 60 disallowed genes in the β-cell14,15, seven of which are present in both lists. (you mentioned more than 60, but in the paper less are mention, should I stick to the numbers of the review?). These lists include a few well described genes, such as hexokinase I, which is "substituted" in β-cells and liver by the much higher Km isoform HK4 (glucokinase) as described before. Interestingly, other genes cluster into the same functional groups, such as those involved in oxidative stress, proliferation or stimulus-secretion coupling and endocytosis (Cite the paper in preparation). The importance of the silence of some of these genes for the β-cell is currently being assessed by different members of the host lab.
Whilst there is abundant published and on-going research focussed to determine the function of those β-cell disallowed genes, only limited information regarding the mechanism of silencing is available.
Historically, the extinction of specific genes was attributed to the action of specific transcriptional repressors or the absence of specific enhancers16 and the study of transcription networks used to attract all the attention. Nonetheless, in the last years, the huge impact that both epigenetic mechanisms (such as DNA methylation or histone modifications) and non-coding RNAs have in gene expression has become evident17,18. Thus, members of the Rutter lab investigated whether DNA methylation or miRNAs contributed to the specific MCT-1 silencing in the β-cell19. Although they concluded that DNA methylation of Slc16a1 promoter does not contribute to its silencing, they found that the β-cell-enriched miRNAs miR-29a/b directly control the expression of Mct-1 thought binding to its 3'UTR.
MiRNAs are a large family -more than a 1000 have been identified so far in humans- of ~22 nucleotides RNAs which regulate virtually every aspect of biology, including development, proliferation, differentiation or metabolism. It is then not surprising that disruption of miRNA function contributes to many human diseases, including cardiovascular disorders, cancer and neurological dysfunctions20.
MicroRNAs are processed from precursor molecules (pri-miRNAs) which are normally transcribed by polymerase II21. Thus, expression of a large subset of mammalian miRNAs may be transcriptionally linked to the expression of other genes, allowing for coordinate regulation of miRNA and protein expression22. The pri-miRNA is first processed in the nucleus by DROSHA and, in mammals, DGCR8 into a ~70nt hairpin, known as pre-miRNA. The pre-miRNA is then exported to the cytoplasm where is further processed by DICER into a small (~20 ntds) RNA duplex. In general, one of the strands (known as the guide) will be incorporated with an Argonaute protein into a miRNA-induced silencing complex (miRISC) while the other one (passenger strand) is released and degraded23. Most metazoan miRNAs direct RISC to target mRNAs by interacting with sites of imperfect complementarity. As a result, the miRNA promotes the degradation and/or inhibit the translation of the target mRNA, resulting in repression of its expression24. In general, the most important region for target recognition comprises the nucleotides 2-8 of the miRNA-known as the "seed" region-and binding sites located in the 3'UTR of the cognate mRNAs are more common25.
A role for miRNAs in pancreatic function was first pointed out by Poy and coworkers in 2004 who identified an islet-specific miRNA, miR-375, that controls insulin secretion26. Later on, a broader role of miRNAs in pancreas development and, more specifically, β-cell function was proved by conditional deletion of Dicer, a necessary enzyme for miRNA maturation. Thus, Dicer was indispensable for pancreas development27, maintaining of the adult pancreas28 and for the development, glucose metabolism and an appropriate insulin-secreting function of the mouse pancreatic β-cells29-31. Over 125 and 200 miRNAs have been detected within the mouse27 and human32 developing pancreas, respectively and altered miRNA expression profile has been found in different mouse models of type II diabetes33. Nevertheless, the specific function of only a small subset of these miRNAs has been assessed in β-cells31,34,35, including miR-29a/b which, as mention before, was proved by the host group and others to play a crucial role in disallowing the expression of Mct-1 in β-cells19,36.
Therefore the aim of this project is to unravel the role of miRNAs in pancreatic β-cell function, focusing on the role of these non-coding RNAs in the silencing of β-cell specific disallowed genes.
Research plan
Assessment of those disallowed genes that may be regulated by miRNAs in β-cells.
Those disallowed genes whose expression is repressed by miRNAs are expected to be up-regulated as a response of DICER depletion.
Four different systems for DICER depletion will be generated/analysed:
Î’-cells islets from Dicer-/- pancreas. Pancreas-specific depletion of Dicer will be achieved by crossing mice carrying conditional knockout alleles of Dicer (Dicer-flox) that will be requested to Dr. Clifford J. Tabin37 with mice expressing Cre recombinase under the control of the Pdx1 promoter (Pdx1-Cre) which are available and have been already successfully used to generate other null mice in the host lab38,39. (Should we ask for the mice before submitting the grant?)
A mouse insulin-secreting cell line, MIN640 where depletion of Dicer will be achieved by RNAi (RNA interference), using commercially available siRNAs against Dicer that will be introduced in the cells by transfection.
Mouse β-cell islets will be isolated and also transfected with siRNAs against Dicer. Transfection efficiency might be low, in which case viral vectors available both commercially and in the host lab41, will be used.
Finally, and more importantly, the DICER depletion will be performed -by the same procedures- in the human pancreatic β-cell line EndoC-βH1. This cell line has been successfully generated just a year ago by the Scharfmann group42 and is the first human cell line that retains many of the characteristics of primary mature β-cells, such as the capability of secreting insulin in response to glucose. It is then an invaluable tool for the study of gene function in human β-cells.
In all cases, the effects of DICER depletion in the expression of the >50 genes that have been described as disallowed will be assessed both at the mRNA level (by reverse transcription followed by real time PCR, RT-qPCR) and, when possible, at the protein level (by using the most adequate/available method for each protein, such as Western Blotting (WB), Immunostaining or ELISA). In addition, for a) and d) total RNA from control or Dicer-depleted cells will be sent to the Genomic Laboratory in Hammersmith (Imperial College core facility) for High-throughput sequencing (need to talk to them first?). By this super-sensitive and accurate method43, we will be able to determine the effects of Dicer deletion not only in the expression of the disallowed genes, but in the whole transcriptome of the pancreatic beta cells.
Those disallowed genes whose expression is up-regulated as a consequence of DICER depletion will be considered as putative miRNA targets, and will be selected for further investigation.
Determination of miRNAs involved in the repression of the selected disallowed genes.
The next stage of the project will aim to determine the specific miRNAs responsible of the silencing of each of the preselected disallowed genes and their mechanism of action. Two main approaches will be undertaken:
In silico studies
The best characterized features that determine miRNA-target recognition are six-nucleotide seed sites, which perfectly complement the 5' end of the miRNA (positions 2-7)25. Also, a match with miRNA nucleotide 8, an A across from nucleotide 1 or both augment the seed pairing and enforce the miRNA-mediated repression44. These seed-pairing rules are widely used to predict functional miRNA target-sites, normally in combination with the secondary structure of the 3'UTR, the neighboring context information or/and the evolutionary conservation25,45,46. Based on that, in the last years several miRNA target prediction programs have been published47,48. We will use few of these bioinformatics tools, such as TargetScan, Miranda o PicTar47, to predict miRNA binding sites present in our candidate genes. These programs typically predict hundreds to thousands of targets for each miRNA, including a high proportion of not bona-fide candidates, but we will focus on those predicted miRNAs that fulfil one of these criteria:
Those miRNAs that had been already shown to have a role in β-cell function or whose expression had been described as altered upon pancreas development and/or animal models of diabetes.
Those miRNAs that are both abundant and β-cell specific (higher expression in beta cells in comparison with other tissues), since both parameters are thought to affect the capacity of a miRNA to interact with its targets. Members of the host lab have generated a matrix representing abundance versus specificity of several miRNAs expressed in β-cells, that allows the selection of those miRNAs with highest combination of abundance and specificity. This approach was successfully used by Pullen and other members of the lab and lead to the discovery of miR-29b as a repressor of Mct-1 in β-cells19.
Those miRNAs whose expression is altered in Ampk-/- mice, available in the host lab as a model of impaired insulin secretion. Members of this lab found that islets from mice specifically inactivated for the two catalytic alpha subunits of AMP-activated protein kinase (Ampk)49 which display defective insulin secretion and glucose homeostasis also exhibit un-regulation of disallowed genes (personal communication from Dr GA Rutter-this isn't published, is it?). Those miRNAs downregulated in this model are therefore candidate regulators of disallowed genes. We will profile miRNAs in comparison with wild type mice by the use of taqman-based low density arrays, commercially available. This real-time PCR-based system allows the assessment of the expression of 641 mouse-specific miRNAs simultaneously by a relatively low price and has been used successfully before -by myself and others- for miRNA expression profiling in multiple and disparate studies32.
In the practice, these bioinformatics tools have a very good performance for prediction of highly conserved and consensus binding sites, but a low efficiency for the detection of non-conserved binding sites as well as for those sites with poor pairing in the seed sequence. Another limitation is that they don't take into account the possibility of tissue specific interaction. Therefore, experimental approaches for miRNA target determination are going to be undertaken in parallel:
Ex vivo studies
An mRNA that is being actively repressed by miRNAs is going to be found in complexes with the repressing miRNA and RISC18. Yoon and colleagues proposed a systematic approach termed MS2-TRAP (tagged RNA affinity purification) for identifying miRNAs associated with a target transcript in the cellular context. Briefly, they tagged the mouse linRNA-p21 with MS2 hairpins and co-expressed it in MEFs along with the chimeric protein MS2-GST. Then they affinity-purified the miRNAs present in the RNP complexes using glutathione-SH beads and those were detected by qPCR. Out of the 5 miRNA analysed (predicted to target linRNA-p21), 4 were enriched in the pulldown and two of them functionally validated 50.
We propose here to use the same approach with one or few of the disallowed genes that we had selected in 1), performing the experiments in MIN6 and/or EndoC-βH1 cells. The identity of the affinity purified miRNAs will be assessed for the miRNAs of interest (selected as in 2a)) by individual RT-qPCR assays and, in a high-throughput manner by taqmann-based arrays or small-RNA ultrasequencing, depending on the quantity of recovered material. This method will allow not only the detection of miRNAs targeting a given disallowed gene, but also proves the direct interaction between the miRNA and the transcript.
The first experiments of this kind-and the optimization of the technique-will be performed using the Acot7 3'UTR. Acot7 is an acyl-CoA thioesterase involved in acyl-CoA hydrolysis and broadly expressed in mouse tissues, while its levels are very low in β-cells14. The disallowance of this gene is relevant for the correct β-cell function, since ectopic expression of this enzyme in (where??) lead to a decrease in the insulin release in response to glucose (Figure 1, I need more data to understand well and explain this, also for the figure legend: which kind of construct?which cells…what is the domain in yellow-required for the effect?)…
Validation of the miRNA-target mRNA interaction.
In order to confirm that the resultant miRNAs are capable of directly targeting the proposed disallowed genes, validation experiments will be performed as follows:
The 3'UTR of the disallowed gene will be cloned downstream of the luciferase ORF (open reading frame) in vectors that will be transfected in both MIN6 and EndoC-βH1 cells, together with inhibitors of the candidate miRNAs or a control. A higher luciferase activity in cells where the miRNA activity is inhibited will be anticipated. In addition, punctual mutations of the sequences identified as miRNA binding sites will be generated in the same reporters. These mutations should disrupt the miRNA-target interaction, supporting the direct effect of the miRNA.
Functional validations will be performed by overexpression/inhibition of the candidate miRNAs in MIN6 cells, EndoC-βH1 cells and/or mouse isolated islets. The specific approach of transfection will be slightly different between the several cell types but will basically consist in: i) transfection of miRNA expression plasmids as in19 or commercially available mimics as in51 and, ii) transfection of commercially available specific LNA (locked nucleic acid) miRNA inhibitors. LNA are a class of high-affinity RNA analogues that exhibit a very high specificity and affinity for RNA and DNA, resulting in a great capacity for inhibition of miRNAs (www.exiqon.com).
The effects of these molecules in the disallowed genes will be determined at the RNA level by RT-qPCR. Often, miRNAs exert their function at the level of translation, in which case alterations in the abundance of the transcripts won't occur. Therefore, changes at the protein level will be also analysed when possible, mainly by WB, immunoblot or ELISA.
Since levels of disallowed genes are low in β-cells - the expression of those genes is already being repressed - it may be, in some cases, difficult to detect a further repression by using miRNA mimics. In those cases, a cell line/model where the target gene is highly express will be selected and used for the miRNA overexpression experiments.
All these kind of validation techniques have been successfully used before by both the host lab and myself19,51, so all the necessary tools will be available and/or relatively easily generated in the host lab.
Assessment of the biological relevance of the studied miRNAs.
Potentially, one miRNA can simultaneously repress different targets with disparate functions. In that case, the phenotype observed after inhibiting a given miRNA won't necessarily be the same than the one observed when the (target) disallowed gene is miss-expressed. Then, importantly, when performing the experiments of miRNA overexpression/inhibition (3b) an assessment of the overall effect in the β-cell function will be carried out. This will be possible thanks to the wide spectrum of techniques that have been developed in the host lab during the years for the analysis of β-cell function. Those techniques include in vitro measurement of insulin secretion, electrophysiological measurements and Ca2+ imagining or transmission electron microscopy. The selection of any of these techniques or others will be done depending on the miRNA to study and the target genes proposed, therefore it is not totally predictable at this point.
Future goals
Although the research plan exposed would be achievable during the duration of the Junior Research Fellowship, this work would potentially open the door to a whole new level of study of the influence of miRNAs in both the normal function of the β-cell and during the development of disease (diabetes).
For example, the specific function of the selected miRNAs could be studied in vivo by generating mice with pancreas-specific miRNA deletion or gain of function. It would be especially interesting to restore the expression of specific miRNAs in the (pancreas-specific) Dicer-/- mice and to compare the two phenotypes. Both approaches would shed more light into the miRNA role and, if miRNAs key for the β-cell function are identified, it could establish the base for the consideration of those miRNAs as therapeutic targets. Several miRNAs have been considered so far for their therapeutic value in different types of disease (Ref.). One of its biggest limitations is the specific delivery of the mimics or inhibitors to the desired tissue; interestingly, one of the open projects of the host lab is aimed to generate cell-specific delivery agents, which could be of grand use in the future in combination with a deeper knowledge of the biology of those small non-coding RNAs in the β-cell.
(I don't remember if that was a project, or a collaboration, or something else…)
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