Functional significance of repressor element 1 silencing transcription factor (REST) target genes in pancreatic beta cells
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The expression of several neuronal genes in pancreatic beta cells is due to the absence of the transcription factor repressor element 1 (RE-1) silencing transcription factor (REST). The identification of these traits and their functional significance in beta cells has only been partly elucidated. Herein, we investigated the biological consequences of a repression of REST target genes by expressing REST in beta cells.
The effect of REST expression on glucose homeostasis, insulin content and release, and beta cell mass was analysed in transgenic mice selectively expressing REST in beta cells. Relevant target genes were identified in INS-1E and primary beta cells expressing REST.
Transgenic mice featuring a beta cell-targeted expression of REST exhibited glucose intolerance and reduced beta cell mass. In primary beta cells, REST repressed several proteins of the exocytotic machinery, including synaptosomal-associated protein (SNAP) 25, synaptotagmin (SYT) IV, SYT VII, SYT IX and complexin II; it impaired first and second phases of insulin secretion. Using RNA interference in INS-1E cells, we showed that SYT IV and SYT VII were implicated in the control of insulin release.
The data document the critical role of REST target genes in pancreatic beta cells. Specifically, we provide evidence that the downregulation of these genes is detrimental for the exocytosis of large dense core vesicles, thus contributing to beta cell dysfunction and impaired glucose homeostasis.
KeywordsExocytosis Insulin secretion RE-1 RE-1 silencing transcription factor REST Synaptotagmins Transgenic mice
β-2-aminobicyclo [2.2.1] heptane-2-carboxylic acid
green fluorescent protein
human growth hormone
intraperitoneal glucose tolerance test
large dense core vesicles
UNC-18 homologue 1
repressor element 1
RE-1 silencing transcription factor
- RIP II
rat insulin II promoter
small interfering RNA
soluble N-ethylmaleimide-sensitive factor attachment protein receptor
sterol regulatory element-binding protein-1
The transcription factor repressor element 1 (RE-1) silencing transcription factor (REST), also known as neuron-restrictive silencing factor, has been implicated in the control of glucose-induced insulin secretion in the βTC3 cell line . This GLI-Kruppel zinc finger transcription factor was first described as a silencer of neuronal genes outside the central nervous system, since its expression is restricted to non-neuronal cells and undifferentiated neural progenitors, allowing genes encoding fundamental neuronal traits to be exclusively expressed in mature neurons [2, 3]. Target genes possess a 21 bp cis element called RE-1 or neuron restrictive silencer element, to which REST binds to inhibit expression. Initial studies indicated that many REST target genes contribute to synaptic plasticity/remodelling, inasmuch as REST regulates the expression of synaptic vesicle proteins , voltage-sensitive ion channels  and neurotransmitter receptors [5, 6]. However, increasing evidence suggests that the significance of the REST/RE-1 system is diverse in embryonic and adult cells and depends on the range of target genes that REST interacts with. Accordingly, a bioinformatic analysis recently revealed about 1,800 putative REST target genes within the human genome, with attributed roles ranging from transcriptional regulation through to metabolism and various aspects of neuronal function . Previous reports have identified some of these target genes and their function in and outside the nervous system. Thus, it is now documented that REST target genes are involved in the reactivation of the fetal cardiac gene programme in hypertrophied and failing hearts , and modulate the vascular plasticity/remodelling of human neointimal hyperplasia .
Pancreatic beta cells, which lack REST , also express a number of REST target genes. However, it remains to be established how these genes participate in beta cell function. Investigating this molecular mechanism, we previously showed that in beta cells REST controls the expression of two neuron-specific genes that code for connexin 36 (CX36), a gap junction-forming protein participating in control of insulin secretion , and islet brain-1, a scaffold protein protecting beta cells against apoptosis via the c-Jun N-terminal kinase signalling pathway . Since the whole set of REST target genes is involved in the control of insulin secretion in vitro , we have now examined the in vivo consequences of a gain of function of REST and have identified additional target genes that are significant to beta cell function. We show that transgenic mice specifically expressing REST in beta cells exhibit glucose intolerance and defective insulin release. We further demonstrate that REST expression affects both triggering and amplifying pathways of insulin secretion, by impairing the expression of proteins of the exocytotic machinery, including the hitherto neglected synaptotagmin (SYT) IV and VII. These data demonstrate the crucial role of REST targets in the control of insulin release and suggest that the downregulation of these genes may contribute to the pathophysiology of beta cell defects.
Pancreases were fixed in 4% (wt/vol.) paraformaldehyde, equilibrated overnight in 15% (wt/vol.) sucrose, embedded in 15% sucrose–7.5% (wt/vol.) gelatin and quickly frozen in methylbutane/liquid nitrogen. Cryosections were incubated overnight at 4°C with either polyclonal rabbit antibodies against human REST or polyclonal guinea pig antibodies against insulin. Primary antibodies were detected using appropriate fluorescein or rhodamine-conjugated antibodies. Sections were viewed on a fluorescence microscope (Leica, Nidau, Switzerland).
Pancreas fragments were fixed in a 2.5% (vol./vol.) glutaraldehyde solution in 0.1 mol/l phosphate buffer (pH 7.4), postfixed in 1% (wt/vol.) osmium tetroxide in the same buffer, dehydrated and embedded in Epon . Thin sections were examined in a Philips CM10 electron microscope (Philips, Eindhoven, the Netherlands).
Glucose tolerance and plasma insulin
Male mice of 12 to 16 weeks were fasted for 14 h before blood samples were collected from the tail vein at 0 (fasting blood sample), 15, 30 and 120 min after an intraperitoneal injection of glucose (2 g/kg body weight as a 20% solution). Blood glucose levels were measured with a Glucometer (Bayer Healthcare, Zurich, Switzerland). Plasma insulin levels from the same time points were determined by ELISA (Mercodia, Uppsala, Sweden).
Cell line and mouse islet isolation
The rat insulinoma cell line INS-1E was maintained in RPMI 1640 medium, as previously described . Islets of Langerhans of adult C57BL/6 male mice, weighing 25 to 30 g, were isolated and cultured as previously described .
INS-1E cells were seeded in 12-well plates and cultured for 48 h. Isolated islets were cultured overnight in non adherent dishes. Cells and islets were infected with adenoviruses as previously described , with a multiplicity of infection of 10 and 15, respectively. As judged by immunofluorescence and quantitative PCR detection of the REST transgene, this procedure typically resulted in the efficient transduction of 70 to 80% INS-1E cells and 30 to 40% primary islet cells. Insulin secretion of INS-1E cells and mouse islets perifusion were assessed in KRB–HEPES (KRBH) supplemented with the indicated stimuli, as published . When 30 mmol/l KCl was added in the experiments testing diazoxide, the NaCl concentration of the medium was reduced to 105 mmol/l. Glucose, leucine, β-2-aminobicyclo [2.2.1] heptane-2-carboxylic acid (BCH), forskolin, 3-isobutyl-1-methylxanthine (IBMX) and diazoxide were purchased from Sigma (Fluka Chemie, Buchs, Switzerland).
Cell and mitochondrial membrane potentials
INS1-E cell and mitochondrial membrane potentials were monitored using 100 nmol/l bis-oxonol and 10 μg/ml rhodamine-123 (Molecular Probes, Eugene, OR, USA), respectively, as previously described .
Cytosolic calcium was monitored in INS-1E cells loaded for 90 min with 2 μmol/l Fura-2AM (Teflab, Austin, TX, USA) in KRBH at 37°C. Ratiometric measurements of Fura-2 fluorescence were performed in a plate-reader fluorimeter (Fluostar Optima; BMG Labtechnologies, Offenburg, Germany) with filters set at 340/380 nm for excitation and 510 nm for emission.
Chromatin immunoprecipitation assay
INS-1E cells were cross-linked with 1% (wt/vol.) formaldehyde for 30 min and the reaction was stopped by addition of 0.125 mol/l glycine. Pelleted cells were lysed in an SDS buffer and submitted to sonication to obtain the desired chromatin length (~500 bp). Chromatin was precleared by addition of blocked protein A Sepharose (Amersham Bioscience Europe, Otelfingen, Switzerland) and the supernatants were immunoprecipitated overnight at 4°C with either polyclonal rabbit antibodies specific to human REST  or to irrelevant sterol regulatory element-binding protein-1 (SREBP-1) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The protein–antibody complexes were collected by addition of protein A Sepharose for 1 h and then washed. The protein–DNA complexes were eluted, treated with RNAseA and proteinase K and then the cross-links were reversed overnight at 65°C. DNA was submitted to PCR amplification of rat RE-1 flanking sequences using specific primers (see Electronic supplementary material [ESM] Table 1).
RNA isolation and northern blotting
RNA isolation and northern blot analysis were performed as previously described . Transcripts levels were revealed using specific probes for rat Cx36 (also known as Gja9), β-actin , rat Munc18-1 (also known as Stxbp1), Syntaxin 1A and Rab3a. For the other genes, probes were obtained with cDNA from INS-1E cells or rat brain, which was amplified with specific primers (ESM Table 2).
Quantitative RT-PCR was performed using a kit (SYBR Premix Ex Taq PCR Kit TaKaRa; Axon Lab, Le Mont-sur-Lausanne, Switzerland) in a Lightcycler (Roche Diagnostics, Mannheim, Germany), as previously described . cDNAs were amplified using specific primers (ESM Table 3).
Western blots were performed as previously described . Specific protein levels were revealed with polyclonal rabbit antibodies against: CX36 , synaptosomal-associated protein (SNAP) 25 (kindly provided by H. Hirling, EPFL, Lausanne, Switzerland) and SYT IV. Monoclonal antibodies against β-actin (Fluka Chemie) were used to normalise the signals.
SYT IV, SYT VII and REST silencing vectors
Specific small interfering RNA (siRNA) were designed as follows: for rat Syt4, the sense target sequence 5′GAAGCACAAAGTGAAAACCA3′ was deduced from the reported mouse sequence ; for rat Syt7 the sense sequence 5′TCATCACCGTCAGCCTTAG3′ was selected according to a previous publication ; for REST, the sense sequence 5′GGAACCTGTTGAGAAGGGA3′ was selected using the siRNA Target Finder (Ambion, Austin, TX, USA). Two complementary DNA fragments encoding the target sequence and separated from the reverse complement by a short spacer were synthesised by Microsynth (Balgach, Switzerland). The two fragments were annealed and cloned downstream of the H1-RNA promoter of the pSuper vector, using BglII–HindIII sites. BamHI–HindIII fragments containing the siRNA constructs were subcloned in a plasmid (pXGH) encoding human growth hormone (hGH).
Human growth hormone secretion
INS-1E cells were transiently transfected with the pXGH vector to use hGH as a reporter for secretion or with the pXGH containing one of the specific siRNAs. At 72 h after transfection, secretion was assessed in KRBH supplemented with 20 mmol/l glucose, 10 μmol/l forskolin and 100 μmol/l IBMX, and measured as published .
Data were expressed as mean ± SD or SEM. Differences between means were assessed using Student’s t test. Statistical significance was defined at a value of p < 0.05, p < 0.01 and p < 0.001.
Transgenic mice featuring a beta cell-targeted expression of REST exhibit reduced insulin content and altered tolerance to glucose
Morphometric analysis of pancreas
Pancreas weight (mg)
Insulin content (pmol/mg pancreas)
Islet numerical density (n per cm2)
Beta cell numerical density (n per cm2)
239 ± 22
26 ± 0.23
62.1 ± 2.6
4,084 ± 483
228 ± 13
13 ± 0.28**
65.7 ± 8.4
2,688 ± 337*
REST target genes are crucial for both first and second phases of insulin secretion
To investigate this impairment, INS-1E-cells were infected with REST-encoding adenovirus (Ad-REST). Compared with control green fluorescent protein (GFP) transduction (Ad-GFP), ectopic REST expression did not alter insulin content (400 ± 60 vs 423 ± 39.6 pmol/l, in GFP and REST-transduced cells, respectively) or basal insulin secretion (ESM Fig. 1). In contrast, it reduced insulin release in response to stimulating concentrations of either glucose, KCl or leucine, compared with GFP-transduced cells (ESM Fig. 1a). These data show that the KATP channel-dependent pathway of insulin secretion, triggered by KCl, as well as the KATP channel-independent pathway, triggered by the non-metabolisable leucine analogue BCH , were both decreased in cells expressing REST. In the diazoxide experiments, the cells, in presence of 250 μmol/l diazoxide and 30 mmol/l KCl, were stimulated with 2.5 mmol/l glucose and then with 15 mmol/l glucose. The second incubation at 15 mmol/l glucose, reflecting the amplifying effect of the sugar on insulin secretion , was significantly reduced in REST-expressing INS-1E cells (ESM Fig. 1a). Similar observations were made in REST and GFP-transduced mouse islets (ESM Fig. 1b), which also featured similar insulin contents (73.8 ± 6.4 vs 66.9 ± 5.4 pmol/l, in GFP and REST-transduced islets, respectively).
REST does not affect plasma and mitochondrial membrane potentials or intracellular levels of Ca2+
To investigate the intracellular signalling leading to altered insulin release of REST-expressing INS-1E cells, we first studied the hyperpolarisation of the mitochondrial membrane, which results from the activation of mitochondrial metabolism. This hyperpolarisation induced by glucose was not affected when cells were infected with Ad-REST (ESM Fig. 2a). We then evaluated the cell membrane depolarisation resulting from the ATP-dependent closure of KATP channels. INS-1E cells expressing REST depolarised normally under glucose stimulation (ESM Fig. 2b). Since membrane depolarisation triggers Ca2+ influx through voltage-gated Ca2+ channels, we measured the cytoplasmic levels of this cation. We found that the expression of REST did not affect the glucose-induced increase in intracellular Ca2+ (ESM Fig. 2c). These data suggest that REST does not alter the intracellular signalling that is activated by mitochondrial metabolism and leads to closure of KATP channels, cell membrane depolarisation and elevation of cytosolic Ca2+.
Identification of functional RE-1 sequences
The in situ binding activity of these RE-1s in REST-expressing INS-1E cells was assessed by a chromatin immunoprecipitation assay. The RE-1s of the Snap25, Syt4, Syt7, Nsf and Cplx2 genes were immunoprecipitated with the antibodies against REST (Fig. 5b), confirming the functional properties of these RE-1 sequences. In contrast the RE-1 of Munc18-1 was not enriched in the cells transduced for REST, consistent with the more variable sequence of this RE-1 motif (Fig. 5a).
REST represses a subset of genes implicated in exocytosis
SYT IV or SYT VII silencing inhibits hormone secretion
Through its loss of function at different stages of neural differentiation, the transcriptional repressor REST plays a strategic role in the specification of the neuronal phenotype, . The absence of REST from mature pancreatic beta cells also assigns to this cell type a specific pattern of genes, termed neuronal traits . Recently, these traits have been implicated in the functioning of a transformed pancreatic beta cell line . However, it has not been elucidated whether any of these genes is directly involved in primary beta cell function and if so, through which mechanism. In the present study, we show that the target genes of REST are crucial for proper function of adult primary beta cells, inasmuch as a gain of REST function impairs first and second phases of insulin secretion. Our data suggest that REST controls a step of the stimulus–secretion coupling pathway, which is downstream of the elevation of intracellular Ca2+ and common to both first and second phases of insulin secretion. We hypothesised that it may be related to vesicle trafficking and/or fusion. Accordingly, we found that REST repressed the expression of several genes coding for proteins of the exocytotic machinery, including SNAP25, SYT IV, SYT VII, SYT IX and complexin II. The exocytosis of insulin-containing LDCV resembles that of neuronal synaptic vesicles  and employs a similar broad set of proteins, each of which plays a precise role. In particular, a direct implication in the control of insulin secretion has been established for several members of the exocytotic machinery, including SNAP25 , NSF , MUNC18–1  and both SYT V and SYT IX . However, the role of SYT IV and SYT VII in neurons and pancreatic beta cells is still a matter of debate. In neuroendocrine PC12 cells, SYT IV has been identified as an essential component for the maturation of secretory granules . This protein has also been reported to play a role in the stabilisation of the fusion pore and in the choice between kiss-and-run and full fusion events, for both synaptic vesicles of PC12 and LDCV of MIN-6 cells [32, 33]. In PC12 cells, SYT VII has also been implicated in the exocytosis of synaptic vesicle [19, 34, 35], whereas in beta cells this protein may be implicated in endocytotic traffic and insulin exocytosis [36, 37]. Using specific siRNAs, we confirm the recently published data on the role of SYT VII in insulin secretion  and show that SYT IV also plays a significant role in the control of insulin release from INS-1E cells.
The integrity of the exocytotic machinery is critical for glucose homeostasis, as observed in the Goto–Kakisaki rat model of type 2 diabetes and in type 2 diabetic patients, in both of which the expression of SNARE proteins is decreased [38, 39]. In view of the present knowledge on granule dynamics and distinct pools, it has been speculated that the proteins of the exocytotic machinery that are implicated in the rate-limiting priming of secretory granules are strong candidates for the triggering of beta cell dysfunctions associated with type 2 diabetes . In the RIP–REST transgenic mice that specifically express REST in beta cells, expression of multiple REST target genes, including those coding for the major proteins controlling exocytosis, was decreased. This model provides a unique opportunity to assess, in vivo, the function of REST-dependent genes. We observed that these genes are critical for insulin secretion, inasmuch as their downregulation resulted in loss of glucose-dependent and glucose-independent insulin release to a degree sufficient to cause glucose intolerance. In these mice, the expression of REST did not alter the architecture of pancreatic islets, but induced a reduction in the number of beta cells. This effect is consistent with the anti-proliferative action of REST, previously reported in tumoral transformation  and human neointimal hyperplasia . The reduced number of cells correlated with reduced expression of the insulin gene and lower pancreatic content of the hormone, which was not observed in INS1-E cells acutely transduced for REST. This lower rate of insulin production may be accounted for by the decreased insulin exocytosis rate and/or the long term REST-induced repression of key factors, such as the protein tyrosine phosphatase, receptor type, N (ICA512). Upon exocytosis, this LDCV-associated protein is cleaved and stimulates insulin synthesis through a retrograde pathway in order to adjust insulin production to its exocytosis . The Ica512 gene is a bona fide RE-1 containing REST target gene, whose expression was decreased in both REST-expressing INS-1E cells and islets of RIP–REST transgenic mice (data not shown).
According to recent data on the genome-wide chromatin occupancy mediated by REST in non neuroendocrine cells, new target genes implicated in different cell functions remain to be found and their effects evaluated [43, 44]. The multiplicity of these genes and their functional interactions make it necessary to perform comprehensive studies using gene array-based analyses of RIP–REST mice. Such studies should identify which of these genes is key to beta cell identity, insulin secretion and beta cell survival. Such a comprehensive analysis was beyond the scope of our study and will be the topic of future experiments.
In summary, using an innovative approach of ectopic REST expression in beta cells, we have identified several target genes that significantly contribute to the in vitro and in vivo control of glucose-stimulated insulin secretion. Our findings document the physiological importance of the native downregulation of REST in beta cells and possibly in other neuroendocrine cells  with a view to maintaining normal secretion by regulating the levels of key exocytotic proteins. Our findings point to the RIP–REST transgenic mice as a pertinent model for the identification of other REST target genes that may play a critical role in the pathophysiology of glucose intolerance and, possibly, of diabetes.
The work of our teams was supported by grants from the Swiss National Science Foundation (31-109530, 31-107644/1, 31-109402, 31-109281/1 and 3200B0-101746), the Juvenile Diabetes Foundation International (1-2005-46 and 1-2007-158), the Placide Nicod Foundation, the Octav and the Marcella Botnar Foundation, the Novartis Foundation, The Emma Muschamp Foundation, The Endocrinology Geneva Foundation, Novo Nordisk and the Geneva Program for Metabolic Diseases.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
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