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Insm1 cooperates with Neurod1 and Foxa2 to maintain mature pancreatic β-cell function.

Jia S, Ivanov A, Blasevic D, Müller T, Purfürst B, Sun W, Chen W, Poy MN, Rajewsky N, Birchmeier C - EMBO J. (2015)

Bottom Line: We defined Insm1, Neurod1 and Foxa2 binding sites associated with genes deregulated in Insm1 mutant β-cells.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.

View Article: PubMed Central - PubMed

Affiliation: Developmental Biology, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany cbirch@mdc-berlin.de jshiqi@mdc-berlin.de.

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Insm1 is expressed in adult pancreatic endocrine cells, and RIPCreER induces efficient and β-cell-specific mutation of Insm1A–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).
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fig01: Insm1 is expressed in adult pancreatic endocrine cells, and RIPCreER induces efficient and β-cell-specific mutation of Insm1A–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).

Mentions: 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 Materials and Methods) 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).


Insm1 cooperates with Neurod1 and Foxa2 to maintain mature pancreatic β-cell function.

Jia S, Ivanov A, Blasevic D, Müller T, Purfürst B, Sun W, Chen W, Poy MN, Rajewsky N, Birchmeier C - EMBO J. (2015)

Insm1 is expressed in adult pancreatic endocrine cells, and RIPCreER induces efficient and β-cell-specific mutation of Insm1A–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).
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fig01: Insm1 is expressed in adult pancreatic endocrine cells, and RIPCreER induces efficient and β-cell-specific mutation of Insm1A–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).
Mentions: 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 Materials and Methods) 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).

Bottom Line: We defined Insm1, Neurod1 and Foxa2 binding sites associated with genes deregulated in Insm1 mutant β-cells.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.

View Article: PubMed Central - PubMed

Affiliation: Developmental Biology, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany cbirch@mdc-berlin.de jshiqi@mdc-berlin.de.

Show MeSH