Identification of Novel Genes Involved in Hyperglycemia in Mice

Abstract

Current attempts to prevent and manage type 2 diabetes have been moderately effective, and a better understanding of the molecular roots of this complex disease is important to develop more successful and precise treatment options. Recently, we initiated the collective diabetes cross, where four mouse inbred strains differing in their diabetes susceptibility were crossed with the obese and diabetes-prone NZO strain and identified the quantitative trait loci (QTL) Nidd13/NZO, a genomic region on chromosome 13 that correlates with hyperglycemia in NZO allele carriers compared to B6 controls. Subsequent analysis of the critical region, harboring 644 genes, included expression studies in pancreatic islets of congenic Nidd13/NZO mice, integration of single-cell data from parental NZO and B6 islets as well as haplotype analysis. Finally, of the five genes (Acot12, S100z, Ankrd55, Rnf180, and Iqgap2) within the polymorphic haplotype block that are differently expressed in islets of B6 compared to NZO mice, we identified the calcium-binding protein S100z gene to affect islet cell proliferation as well as apoptosis when overexpressed in MIN6 cells. In summary, we define S100z as the most striking gene to be causal for the diabetes QTL Nidd13/NZO by affecting β-cell proliferation and apoptosis. Thus, S100z is an entirely novel diabetes gene regulating islet cell function.

Keywords: QTL; apoptosis; diabetes; proliferation; β-cell.

Figure 1

Physical maps of the QTL Nidd13/NZO identified by genome-wide linkage analysis of NZO backcross populations. Genome-wide linkage analysis for the traits blood glucose (A) and total pancreatic insulin (B) revealed a single QTL on chromosome 13. The analysis was performed with data from male mice of the different N2 populations. The horizontal line indicates the threshold of significance (p < 0.01). (C) Effect sizes of traits associated with the QTL Nidd13/NZO in the (NZOxB6)N2 cohort (N/B: n = 148; N/N: n = 139). Data are presented as means ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.005.

Figure 2

Phenotypic traits of congenic Nidd13/NZO mice. Male mice carrying 42.4 Mbp of chromosome 13 from NZO or B6 (N/N, N/B, B/B) on the NZO background were characterized for the development of body weight, body fat, and blood glucose on high-fat diet until 10 weeks of age. Data are presented as means ± SEM. * p < 0.05; B/B, n = 8–11; N/B, n = 31–33; N/N, n = 8–11.

Figure 3

Combined approach of haplotype and gene expression analysis in islets for the identification of gene variants within the Nidd13/NZO locus. (A) Haplotype map of single nucleotide polymorphisms (SNPs) within the critical region of Nidd13/NZO (78.0–120.4 Mbp). Black line: total number of SNPs (all SNPs annotated for the B6 reference genome). Red line: SNPs according to B6≠NZO. (B) Genes within the critical Nidd13/NZO interval displaying a differential expression in pancreatic islets of parental mice, (C) Nidd13/NZO.42.2 congenics, and (D) B6-ob/ob versus NZO mice. (E) Single-cell data of Nidd13/NZO candidate genes. Dot plot representing expression levels by color code and fraction of cells expressing the gene. Expression levels are depicted from white to red (low to high), and dot size presents fraction of cells expressing the indicated gene. * p < 0.05; ** p < 0.005.

Figure 4

Impact of the different Nidd13/NZO candidate genes on proliferation. (A) MIN6 were infected with an empty adenovirus (Ad-∅) as control or an adenovirus expressing one of the Nidd13/NZO candidate genes; overexpression was analyzed by qRT-PCR. (B) Infected MIN6 cells were incubated with BrdU for 72 h. For quantification, cells were counted and assessed as BrdU positive or negative. For each group, 10 pictures of at least two coverslips per infection (n = 4) were evaluated. (C) Representative immunocytochemical co-stainings of BrdU (magenta) and DAPI (blue). (D) Primary islets of NZO mice were dispersed and infected with an empty adenovirus (Ad-∅) as control or an adenovirus expressing S100z, and overexpression was analyzed by qRT-PCR. After incubation with BrdU for 48 h, islets were co-stained with a BrdU (magenta) and DAPI (blue) antibody. For each group, at least 10 images of four or two coverslips per infection (n = 2) were evaluated. * p < 0.05.

Figure 5

Effect of Nidd13/NZO candidate genes on apoptosis. Representative images displaying the effect of MIN6 cells infected with (A) Ad-S100z vs. Ad-∅100 and (B) Ad-Ankrd55, Ad-Rnf180, and Ad-Iqgap2 vs. Ad-∅1000 on protein levels of cleaved caspase-3 as a marker for apoptosis. Infected cells (n = 6–8) were stressed with 0.4 mM palmitate for 48 h, and protein levels of cleaved caspase-3 were analyzed and quantified by Western blotting. Here, α-tubulin was used as loading control. (C) Primary islet cells of NZO mice were infected with Ad-S100z or Ad-∅100 and incubated with 0.4 mM palmitate for 48 h. Expression of ER-stress and apoptotic marker genes were analyzed by qRT-PCR. Data are presented as fold change compared to cells under basal conditions. Data are shown as means ± SEM. * p < 0.05; ** p < 0.005, *** p < 0.001.