Insm1 cooperates with N eurod1 and F oxa2 to maintain mature pancreatic β‐cell function
2015; Springer Nature; Volume: 34; Issue: 10 Linguagem: Inglês
10.15252/embj.201490819
ISSN1460-2075
AutoresShiqi Jia, Andranik Ivanov, Dinko Blasevic, Thomas Müller, Bettina Purfürst, Wei Sun, Wei Chen, Matthew N. Poy, Nikolaus Rajewsky, Carmen Birchmeier,
Tópico(s)Diabetes Management and Research
ResumoArticle31 March 2015Open Access Source Data Insm1 cooperates with Neurod1 and Foxa2 to maintain mature pancreatic β-cell function Shiqi Jia Corresponding Author Shiqi Jia Developmental Biology, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Andranik Ivanov Andranik Ivanov Systems Biology of Gene Regulatory Elements, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Dinko Blasevic Dinko Blasevic Developmental Biology, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Thomas Müller Thomas Müller Developmental Biology, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Bettina Purfürst Bettina Purfürst Electron Microscopy Platform, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Wei Sun Wei Sun Scientific Genomics Platform, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Wei Chen Wei Chen Scientific Genomics Platform, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Matthew N Poy Matthew N Poy Molecular Mechanisms of Metabolic Disease, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Nikolaus Rajewsky Nikolaus Rajewsky Systems Biology of Gene Regulatory Elements, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Carmen Birchmeier Corresponding Author Carmen Birchmeier Developmental Biology, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Shiqi Jia Corresponding Author Shiqi Jia Developmental Biology, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Andranik Ivanov Andranik Ivanov Systems Biology of Gene Regulatory Elements, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Dinko Blasevic Dinko Blasevic Developmental Biology, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Thomas Müller Thomas Müller Developmental Biology, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Bettina Purfürst Bettina Purfürst Electron Microscopy Platform, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Wei Sun Wei Sun Scientific Genomics Platform, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Wei Chen Wei Chen Scientific Genomics Platform, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Matthew N Poy Matthew N Poy Molecular Mechanisms of Metabolic Disease, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Nikolaus Rajewsky Nikolaus Rajewsky Systems Biology of Gene Regulatory Elements, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Carmen Birchmeier Corresponding Author Carmen Birchmeier Developmental Biology, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Author Information Shiqi Jia 1,‡, Andranik Ivanov2,‡, Dinko Blasevic1, Thomas Müller1, Bettina Purfürst3, Wei Sun4,6, Wei Chen4, Matthew N Poy5, Nikolaus Rajewsky2 and Carmen Birchmeier 1 1Developmental Biology, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany 2Systems Biology of Gene Regulatory Elements, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany 3Electron Microscopy Platform, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany 4Scientific Genomics Platform, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany 5Molecular Mechanisms of Metabolic Disease, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany 6Present address: Department of Medical Oncology, Jiangsu Provincial Hospital of TCM, Nanjing, China ‡These authors contributed equally to this work *Corresponding author. Tel: +49 30 9406 2403; Fax: +49 30 9406 3765; E-mail: [email protected] *Corresponding author. Tel: +49 30 9406 3848; Fax: +49 30 9406 3765; E-mail: [email protected] The EMBO Journal (2015)34:1417-1433https://doi.org/10.15252/embj.201490819 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Key transcription factors control the gene expression program in mature pancreatic β-cells, but their integration into regulatory networks is little understood. Here, we show that Insm1, Neurod1 and Foxa2 directly interact and together bind regulatory sequences in the genome of mature pancreatic β-cells. We used Insm1 ablation in mature β-cells in mice and found pronounced deficits in insulin secretion and gene expression. Insm1-dependent genes identified previously in developing β-cells markedly differ from the ones identified in the adult. In particular, adult mutant β-cells resemble immature β-cells of newborn mice in gene expression and functional properties. We defined Insm1, Neurod1 and Foxa2 binding sites associated with genes deregulated in Insm1 mutant β-cells. Remarkably, combinatorial binding of Insm1, Neurod1 and Foxa2 but not binding of Insm1 alone explained a significant fraction of gene expression changes. Human genomic sequences corresponding to the murine sites occupied by Insm1/Neurod1/Foxa2 were enriched in single nucleotide polymorphisms associated with glycolytic traits. Thus, our data explain part of the mechanisms by which β-cells maintain maturity: Combinatorial Insm1/Neurod1/Foxa2 binding identifies regulatory sequences that maintain the mature gene expression program in β-cells, and disruption of this network results in functional failure. Synopsis The functional and molecular characterization of Insm1 reveals its crucial role in the maintenance of adult pancreatic β-cell identity. Deletion of Insm1 in adult pancreatic β-cells in mice translates into deficits of insulin secretion. Insm1/Neurod1/Foxa2 co-occupy regulatory sequences to maintain a mature gene expression profile in pancreatic β-cells. Corresponding human Insm1/Neurod1/Foxa2 binding regions show sequence variations that have been implicated in β-cell dysfunction(s). Introduction During terminal differentiation of cells, gene expression programs are established that are then faithfully maintained throughout the lifetime of mature cells (Holmberg & Perlmann, 2012). The ontogeny of pancreatic β-cells, the insulin-secreting cells of the body, has been studied extensively, and a hierarchically controlled transcription factor network that determines β-cell specification and differentiation has been defined. Frequently, these transcription factors provide transient regulatory input, but a subset of them remains expressed during the lifetime of mature non-dividing β-cells (Holmberg & Perlmann, 2012; Szabat et al, 2012). In particular, Neurod1, Foxa1, Foxa2, Nkx6.1 and Pdx1 were first identified to control β-cell development but are now known to also maintain mature β-cell function (Naya et al, 1997; Ahlgren et al, 1998; Holland et al, 2002; Gao et al, 2007, 2010; Gu et al, 2010; Szabat et al, 2012; Taylor et al, 2013). This shows that the persistent and mature gene expression program in β-cells is actively controlled, but only recently the integration of these factors into regulatory networks is beginning to receive attention (Pasquali et al, 2014). Maturity is known to be important for β-cell function, and loss of maturity was associated with failure of glucose-stimulated insulin secretion in diabetes (Weir & Bonner-Weir, 2004; Ziv et al, 2013; Wang et al, 2014). Insulin release is finely tuned and relies on mechanisms that allow β-cells to sense and metabolize glucose, as well as on signaling cascades that couple metabolic signals to insulin exocytosis (Lang, 1999; MacDonald et al, 2005). In addition to glucose as primary regulator, metabolites such as free amino acids or fatty acids also stimulate insulin secretion. Furthermore, hormones such as glucagon-like peptide-1 (Glp1) and gastrointestinal inhibitory polypeptide (GIP) modulate and serve as potentiators of glucose-induced insulin secretion (MacDonald et al, 2005; Baggio & Drucker, 2007). Thus, in a normal physiological setting in the adult, insulin secretion is controlled by glucose and additional signals that provide modulatory input. β-cells of adult and newborn mice differ in their response to these complex cues, indicating that the ability to appropriately secrete insulin is acquired during postnatal maturation. To restore β-cells or β-cell functions in diabetic patients, in vitro protocols were developed that allow the generation of β-cells from embryonic stem cells by a step-wise differentiation that recapitulates development in vivo (D'Amour et al, 2006; Nostro & Keller, 2012). Until recently, such methods yielded immature β-cells that poorly secrete insulin in response to glucose, but a new protocol was recently reported to overcome this limitation (Pagliuca et al, 2014). Despite the physiological importance of β-cell maturity, the maturation process remains little understood on a molecular level. Insm1 encodes a zinc finger factor that controls differentiation of β-cells and other endocrine cell types in the pancreas, intestine, pituitary and adrenal medulla (Gierl et al, 2006; Wildner et al, 2008; Welcker et al, 2013; Osipovich et al, 2014). In endocrine cells of the pancreas, Insm1 expression is initiated early during development in an Ngn3-dependent manner (Gierl et al, 2006; Mellitzer et al, 2006). Insm1 but not Ngn3 expression is maintained in mature endocrine cells, providing an example of the distinct regulatory cascades operative in development and maturity. We show here that Insm1 binds to chromatin in β-cells and that most Insm1 sites are co-occupied by two key β-cell transcription factors, Neurod1 and Foxa2. Using conditional gene ablation in mice, we show that Insm1 controls mature β-cell function and is required for correct glucose-stimulated insulin secretion. Mutant β-cells shift their functional properties and gene expression program to resemble immature β-cells. Binding sites co-occupied by Insm1/Neurod1/Foxa2 are mainly located in intergenic and intronic sequences. Remarkably, the presence of such combinatorial binding sites correlates very significantly with gene expression changes in Insm1 mutant β-cells. Conversely, sites occupied by Insm1 only are enriched in promoters and correlate poorly with gene expression changes. Together, our data provide evidence that combinatorial binding of Insm1, Neurod1 and Foxa2 identifies cis-regulatory sequences that maintain the mature gene expression program of β-cells. Results Insm1 ablation in mature β-cells abrogates glucose-stimulated insulin secretion In the mature murine pancreas of control mice, nuclear Insm1 protein was detected by immunohistology in insulin+ β-cells (Fig 1A and B). Other endocrine cell types like α-cells that express glucagon (Gcg), δ-cells that express somatostatin (Sst) and Pp-cells that express pancreatic polypeptide (Pp) locate to the periphery of murine islets and also express Insm1 (Fig 1C–E). In contrast to the expression observed in developing endocrine cells, Insm1 protein levels in adult endocrine cells appeared heterogeneous and β-cells that express high and low Insm1 levels were observed. To assess the role of Insm1 in mature β-cell gene regulation and function, we introduced a somatic Insm1 mutation in mice using a 'floxed' allele (Insm1flox/lacZ; Supplementary Fig S1A and 4) and a tamoxifen-inducible Cre driven by the insulin promoter (RIPCreER; Dor et al, 2004). To test for specificity of recombination using RIPCreER, the mT/mG indicator line was used that expresses membrane-bound tomato and GFP before and after Cre-dependent recombination, respectively (Muzumdar et al, 2007). After tamoxifen treatment of mT/mG;RIPCreER animals, the vast majority of insulin+ cells co-expressed GFP, indicating that recombination is efficient in β-cells (Fig 1F and G). Non-recombined tomato+ cells in islets were also observed (Fig 1H). These co-expressed PECAM, glucagon, pancreatic polypeptide and somatostatin (Fig 1I–L), indicating that recombination does not occur in endothelial, α-, δ- and Pp-cells. We used tamoxifen-treated mice with an Insm1flox/lacZ;RIPCreER genotype as conditional mutants, which are subsequently called coInsm1 mutants. In coInsm1 mutants, a pronounced reduction of Insm1 protein was observed by immunohistology in insulin+/Pdx1high β-cells (Fig 1M–P) and by Western blot analysis of isolated islets (Fig 2A). Figure 1. Insm1 is expressed in adult pancreatic endocrine cells, and RIPCreER induces efficient and β-cell-specific mutation of Insm1 A–E. Analysis of Insm1 protein (red) in pancreata of adult control mice by immunohistology, using DAPI (blue) as counterstain. Insm1 is present in (A, B) β-cells that express insulin (Ins, green), (C) α-cells that express glucagon (Gcg, green), (D) δ-cells that express somatostatin (Sst, green) and (E) Pp-cells that express pancreatic polypeptide (Pp, green). Arrowheads indicate cells co-expressing Insm1 and Gcg (C), Insm1 and Sst (D), Insm1 and Pp (E). F–L. Analysis of RIPCreER-induced recombination using mT/mG reporter mice that express membrane-bound tomato and GFP before and after Cre-mediated recombination, respectively. The insulin+ β-cells co-express GFP and have thus undergone recombination. Cells that express tomato co-express PECAM, glucagon, pancreatic polypeptide or somatostatin and are thus not recombined. M, N. Co-localization of Insm1 and Pdx1 in nuclei of insulin+ β-cells in control mice. O, P. Insm1 protein is lost in most β-cells of coInsm1 mice. Arrowheads indicate remaining un-recombined β-cells that continue to express Insm1; the open arrowhead points towards an Insm1+ endocrine cell that does not co-express insulin. Data information: Scale bars: 50 μm (A, F, H); 25 μm (E, G, L, P). Download figure Download PowerPoint Figure 2. Conditional mutation of Insm1 results in disrupted glucose-stimulated insulin secretion and glucose intolerance Western blot analysis of Insm1 in isolated islets of control and coInsm1 mutant mice (pool of 200 islets from 2 to 3 mice). Glucose-stimulated insulin secretion in control and coInsm1 mice. The mice were injected with glucose at t = 0, and insulin secretion was monitored over time (n = 12–14). Blood insulin levels in fasted and randomly fed control (green bars: Insm1flox/lacZ; blue bars: Insm1flox/+;RIPCreER) and coInsm1 mice (red bars: Insm1flox/lacZ;RIPCreER) (n = 14–16). Blood glucose levels in fasted and randomly fed control and coInsm1 mice; mutations were introduced at an age of 4–5 weeks, and mice were analyzed at 8–12 weeks. Glucose levels in mutant mice were 12.3 ± 1.3 mM (range of 4.0–24.7 mM), in contrast to 7.5 ± 0.2 mM and 7.2 ± 0.2 mM (range of 6.0–10.2 mM) in control mice (n = 12–14). Glucose tolerance test in control and coInsm1 mice (n = 12–14). Blood glucose levels in randomly fed control and coInsm1 mice at various time points (0–11 weeks) after introduction of the mutations (n = 12–14). Data information: Data are presented as means ± SEM, statistical significance was assessed by ANOVA and 2-tailed unpaired Student's t-test. *P < 0.05; **P < 0.01; ***P < 0.001. Source data are available online for this figure. Source Data for Figure 2 [embj201490819-sup-0016-SDataFig2.zip] Download figure Download PowerPoint We next assessed plasma insulin levels in control and mutant mice. When insulin secretion was monitored after glucose injection in coInsm1 mutants, we observed a completely disrupted insulin secretion; that is, insulin levels were not elevated 2, 5 or 15 min after the injection (Fig 2B). Insulin secretion was also impaired during a 2-h period after glucose challenge (Supplementary Fig S1B). In fasted coInsm1 and control animals, insulin levels were similar (Fig 2C). We also monitored insulin levels in randomly fed animals that receive a mixture of nutrients, that is, under conditions in which insulin secretion is regulated by complex physiological parameters encompassing glucose, other nutrients and hormones. This showed that insulin levels were increased in randomly fed compared to fasted coInsm1 mice (Fig 2C). However, insulin levels did not reach those observed in randomly fed control mice (Fig 2C). Blood glucose levels of randomly fed coInsm1 animals were markedly elevated, and coInsm1 mice showed pronounced elevation of blood glucose in glucose tolerance tests (Fig 2D and E). Elevated glucose levels were consistently observed in coInsm1 mice 2–11 weeks after tamoxifen treatment (Fig 2F). Similar changes in glucose levels were observed in randomly fed and glucose challenged mice regardless whether the mutation was introduced at an age of 1 or 2 months (compare Fig 2D and E; Supplementary Fig S1C and D). In all experiments described above, we compared coInsm1 mutants to control animals of two genotypes, Insm1flox/+;RIPCreER and Insm1flox/lacZ. Since the two control groups were indistinguishable, we combined them in subsequent experiments. Insm1 is required for normal islet morphology and insulin content We next tested whether decreased insulin release was caused by β-cell loss or reduced insulin production. Pancreatic β-cell numbers were comparable in control and mutant mice (Fig 3A), but β-cell mass was mildly reduced in the mutants (Fig 3B). Insulin content and insulin-1 (Ins1) but not insulin-2 (Ins2) mRNAs were mildly reduced in coInsm1 mutant pancreata (Fig 3C and D). Mice that lack the insulin-1 gene remain glucose tolerant (Leroux et al, 2001), indicating that the mild downregulation of insulin-1 mRNA cannot account for the pronounced deficit in insulin secretion observed in coInsm1 mutant mice. Figure 3. Altered islet morphology in coInsm1 mutant mice A, B. Comparison of β-cell number (A) and β-cell mass (B) in control and coInsm1 mice (n = 3 mice, 6–8 slides/animal). The number given in (A) refers to cell numbers/pancreas area. C, D. Total pancreatic level of insulin (n = 6–12) (C) and Ins1 and Ins2 mRNA in control and coInsm1 mice (n = 5) (D). E. Epithelial morphology and quantification of the packing density of islet cells in control and coInsm1 mice. The number given refers to cell numbers/islet area. F. Analysis of cell death in β-cells of control and coInsm1 mice by TUNEL staining. G. Comparison of proliferation in islets of control and coInsm1 mice using BrdU incorporation. H. Distribution and quantification of glucagon+ cells in pancreata of control and coInsm1 mice. Data information: Data are presented as means ± SD; statistical significance was assessed by ANOVA and 2-tailed unpaired Student's t-test. *P < 0.05; **P < 0.01. (A, B, E–H) n = 3. Scale bars: 20 μm (E); 10 μm (F); 50 μm (G, H). Download figure Download PowerPoint Histological analyses showed that the average cell size was reduced resulting in denser nuclear packing, but overall epithelial cell morphology was intact as assessed by β-catenin distribution (Fig 3E). The smaller cell size can thus account for the reduction in the β-cell mass. TUNEL staining showed that apoptosis rates were elevated in islets of coInsm1 mutants, but they nevertheless remained low (< 1 apoptotic cell/section; Fig 3F). This was accompanied by a mild increase in proliferation (Fig 3G). We also found that glucagon-expressing α-cells were increased in number and dispersed throughout the islet instead of being located in the periphery (Fig 3H). The numbers and locations of somatostatin- and pancreatic peptide-expressing cells and expression of other hormones were unchanged (Supplementary Fig S2). In conclusion, ablation of Insm1 in adult β-cells resulted in perturbed islet morphology, reduced β-cell mass due to smaller cell volume and decreased pancreatic insulin content. Insm1 mutant β-cells assume immature functional characteristics We next tested systematically for changes in gene expression in coInsm1 mutant islets by microarray analysis (GSE54044), which revealed deregulated genes in mutant islets (P-value < 0.05; 1,232 genes with FC > 1.2 and < 0.8; 352 with FC > 1.4 and < 0.6). Similar numbers of genes were up- and down-regulated. To define affected cellular processes, we performed Gene Ontology (GO) term analysis of differentially expressed genes. Consistent with glucose intolerance, deregulated genes were associated with biological processes critical for β-cell function, such as regulation of insulin secretion, response to hormone and glucose stimulus, and glucose metabolism (Fig 4A; Supplementary Table S1). Comparison with Insm1-dependent genes identified previously in the developing pancreas (Gierl et al, 2006) revealed little overlap (61 overlapping genes, Pearson's coefficient 0.21, Supplementary Table S2). Thus, Insm1 controls distinct sets of genes in developing and mature β-cells. Figure 4. Mutation of Insm1 disrupts mature gene expression in adult islets GO term analysis of differentially expressed genes identified by microarray analysis in isolated islets from control and coInsm1 mutant mice. Shown are GO terms, the P-value for their enrichment and the number of differentially expressed genes associated with a particular GO term. Verification of differential expression of genes previously implicated in the control of insulin secretion; RNA from isolated islets of control and coInsm1 mutant mice was compared by qRT–PCR (n = 5). Comparison of the deregulated genes identified in coInsm1 mutant versus control islets and sorted β-cells from P1 and adult mice. 1,232 genes were differentially expressed in coInsm1 mutant islets, and among these, 358 were previously identified as differentially expressed in immature versus mature β-cells. Analysis of Ucn3, Glut2 and Glp1r protein by Western blotting; a representative of three experiments is shown. Insulin secretion from isolated islets of control and coInsm1 mutant mice in response to glucose (3.3 and 16.7 mM), and in response to 16.7 mM glucose and additional secretagogues, that is, 100 mM pyruvate (Pyr), 20 nM exendin-4 (Ex-4), 100 nM gastric inhibitory polypeptide (GIP), 200 μM of the ATP-sensitive K+ channel inhibitor tolbutamide (Tol) and in response to membrane depolarization (30 mM KCl) (n = 3–4). Insulin secretion from isolated islets of P1 and adult mice in response to glucose, secretagogues and membrane depolarization (n = 3–4). Secretory vesicle appearance and density in control and coInsm1 mutant β-cells (n = 3). Arginine-induced insulin secretion was intact in coInsm1 mutant mice (n = 6–15). Data information: Data are presented as means ± SD; statistical significance was assessed by ANOVA and 2-tailed unpaired Student's t-test. *P < 0.05; **P < 0.01; ***P < 0.001. Scale bar: 1 μm. Source data are available online for this figure. Source Data for Figure 4 [embj201490819-sup-0017-SDataFig4.zip] Download figure Download PowerPoint Insulin exocytosis from pancreatic β-cells is stimulated by glucose metabolism by a mechanism involving ATP-sensitive K+ channels (MacDonald et al, 2005). In addition, hormones such as glucagon-like peptide-1 (Glp1) and gastrointestinal inhibitory polypeptide (GIP) and metabolic fuels potentiate insulin secretion (Baggio & Drucker, 2007). Many deregulated genes identified participate in glucose-dependent insulin secretion, such as Glut2 (Slc2a2), Pcx, Pfkfb3, Hk1, Pdk1/4 and G6pc2 that control glucose uptake and metabolism, a subunit of the ATP-sensitive potassium channel (Abcc8), intracellular signaling molecules (Prckb, Ak5, Trpm5, Cx36), glucagon-like peptide-1 receptor (Glp1r) and a negative regulator of GIP receptor signaling (Rgs2) (see Supplementary Table S1 for a list of deregulated genes implicated in insulin secretion; Babenko et al, 2006; Becker et al, 1994; Carvalho et al, 2010; Colsoul et al, 2010; Guillam et al, 1997; Stanojevic et al, 2008; Sugden & Holness, 2013; Tseng & Zhang, 1998; Wang et al, 2007; Xu et al, 2008). The expression of deregulated genes implicated in insulin secretion was confirmed by qRT–PCR (Fig 4B). Ucn3 provides a marker for β-cell maturity, and the ratio of MafB and MafA expression is important during β-cell maturation (Artner et al, 2010; Blum et al, 2012; van der Meulen et al, 2012; Hang et al, 2014). We also observed that Ucn3, MafB and MafA were deregulated in coInsm1 mutant islets. This provided first evidence that coInsm1 mutant islets have lost a mature gene expression program. We therefore systematically compared genes deregulated in coInsm1 islets to previously reported genes differentially expressed in mature and immature (P1) murine β-cells (Blum et al, 2012; GSE35906). This revealed a large overlap; that is, 29% of the genes that were differentially expressed in control and coInsm1 mutant islets were also differentially expressed in mature and immature β-cells; a hypergeometric probability test demonstrated that this overlap was highly significant (P < 5.6 × 10−9; Fig 4C). We also compared the differentially expressed genes of coInsm1 mutants with a diabetes model (Kluth et al, 2014) and found a more limited overlap (115 genes deregulated in both models, P = 1). Islets of coInsm1 mutants resemble thus more closely immature than diabetic islets. Ucn3, Glut2 and Glp1r were among the common set of deregulated genes in immature and coInsm1 mutant islets, and Western blotting demonstrated reduced levels of the corresponding proteins (Fig 4D). Ngn3 and other markers of β-cell progenitors were not induced (Supplementary Fig S3). This indicates that coInsm1 mutant β-cells revert to an immature but not progenitor state. Immature β-cells, for instance those from newborn mice, are known to respond aberrantly to glucose (Rorsman et al, 1989; Blum et al, 2012). We therefore characterized and directly compared glucose-induced insulin secretion from islets isolated from coInsm1 mutants and immature mice (Fig 4E and F). coInsm1 mutant but not control islets released insulin at 3.3 mM glucose, but glucose-stimulated insulin release was similar at 16.7 mM. In control islets, insulin secretion was further enhanced by pyruvate, exendin-4 and GIP in the presence of 16.7 mM glucose, but coInsm1 mutant islets responded poorly to these secretagogues (Fig 4E). Similar changes in insulin secretion were observed in immature islets obtained from postnatal day 1 (P1) mice, that is, increased insulin release at low glucose (3.3 mM) and poor response to pyruvate, exendin-4 and GIP (Fig 4F). coInsm1 mutant islets and immature islets also displayed a similar decrease in sensitivity to tolbutamide, a blocker of ATP-sensitive potassium channels (Fig 4E and F). Stimulation with KCl released similar amounts of insulin from control, coInsm1 mutant and from immature islets (Fig 4E and F). This indicated that the overall secretory machinery remained functional in coInsm1 and immature islets. This was also supported by electron micrograph analysis of insulin-containing secretory granules in β-cells that did not reveal changes in vesicular morphology or density in mutant mice (Fig 4G). Furthermore, examination of arginine-induced insulin secretion in vivo did not reveal significant differences between control and coInsm1 animals (Fig 4H). Thus, mutant β-cells remain fully capable of insulin secretion after challenge with arginine in vivo or by global membrane depolarization by exogenous KCl in vitro. In summary, the primary deficit in coInsm1 mutant β-cells is restricted to glucose-induced insulin secretion and its modulation by secretagogues, excluding a general deficit in the basic secretory machinery. This is also supported by the fact that genes controlling glucose sensing and metabolism, secretagogue response and intracellular signaling are deregulated. Insm1 binds to chromatin cooperatively with Neurod1 and Foxa2 To understand the molecular mechanism of Insm1 function in β-cells, ChIP-seq experiments were performed. As a chromatin source, we used early passages of an immortalized pancreatic β-cell line (referred to as SJ β-cells) that we established (Radvanyi et al, 1993). Insulin secretion from SJ β-cells was stimulated eightfold to tenfold in response to glucose, which is comparable to the response observed in dissociated cells from adult islets, but lower than the one in intact islets, and these cells respond to exendin-4 and GIP (Supplementary Fig S4A; compare with Blum et al, 2012; Halban et al, 1982). Two independent ChIP-seq experiments (GSE54046) identified 17,453 high confidence Insm1 binding sites in chromatin of SJ β-cells that overlapped between experiments (see Supplementary Fig S4B–D for replicate comparisons, antibody specificity and binding site distribution). Example traces are shown in Fig 5A and Supplementary Fig S4E (upper traces). Examination of these binding sites by de novo motif analyses revealed an enrichment of two sequences within ±25 bp of the summits of binding sites (Fig 5Bi and ii; E-value = 8 × 10−92 and 8 × 10−41 for the two motifs shown in i and ii, respectively). The first corresponds to an E-box (present in 22% of all Insm1 binding sites) and the
Referência(s)