Genetic predisposition for beta cell fragility underlies type 1 and type 2 diabetes

Abstract

Type 1 (T1D) and type 2 (T2D) diabetes share pathophysiological characteristics, yet mechanistic links have remained elusive. T1D results from autoimmune destruction of pancreatic beta cells, whereas beta cell failure in T2D is delayed and progressive. Here we find a new genetic component of diabetes susceptibility in T1D non-obese diabetic (NOD) mice, identifying immune-independent beta cell fragility. Genetic variation in Xrcc4 and Glis3 alters the response of NOD beta cells to unfolded protein stress, enhancing the apoptotic and senescent fates. The same transcriptional relationships were observed in human islets, demonstrating the role of beta cell fragility in genetic predisposition to diabetes.

Figure 1

NOD mouse susceptibility to immune-independent diabetes demonstrated through a sensitized transgenic model. (a) Incidence of diabetes in male insHEL transgenic mice on the B10^k (n = 22), NOD^k (n = 43) and (B10^k × NOD^k)F1 (n = 16) backgrounds. No diabetes was observed in non-transgenic male littermates. (b) Incidence of diabetes in male insHEL transgenic mice on the B10^k (n = 22), B10^k.Aire^−/− (n = 11) and B10^k.Aire^−/−.Rag1^−/− (n = 23) backgrounds. B10^k.Aire^−/− mice without the insHEL transgene did not develop diabetes (n = 22). (c) Average number of islets per pancreatic section in B10^k, B10^k.insHEL, NOD^k and NOD^k.insHEL mice at 28 weeks of age (n = 4–5 mice/group; WT, wild type). Data are shown as means ± s.e.m. (d) Incidence of diabetes in male insHEL transgenic mice on the NOD^k (n = 43) and NOD^k.scid (n = 9) backgrounds. No diabetes was observed in nontransgenic male littermates. (e) Diabetes incidence in male insHEL transgenic mice on the B10^k.Rag1^−/− (n = 58), NOD^k. Rag1^−/− (n = 44) and (B10^k × NOD^k)F1. Rag1^−/− (n = 51) backgrounds. (f) Hematoxylin and eosin histology of pancreatic islets at 28 weeks of age (representative of 7–15 mice/group). Scale bars, 50 μm. (g) B10^k.insHEL mice and NOD^k.insHEL mice were irradiated and reconstituted with NOD^k or B10^k bone marrow, respectively, before aging for diabetes incidence (n = 7 and 6). *P < 0.05, **P < 0.001, ***P < 0.0001.

Figure 2

Transgene-induced beta cell stress results in disturbed insulin processing and glucose intolerance. (a)Electron microscopy of beta cells from wild-type and insHEL transgenic B10^k.Rag1^−/− and NOD^k.Rag1^−/− mice at 12 weeks of age was used to assess the number of insulin granules per cellular cross-section (n = 3 mice/group). (b)Electron microscopy images of the cells described in a. Images are representative of three mice per group. Scale bars, 1 μm. (c–f) Fasting serum samples from B10^k.Rag1^−/−, B10^k.Rag1^−/−.insHEL, NOD^k.Rag1^−/− and NOD^k.Rag1^−/−. insHEL mice at 24 weeks of age were assessed by ELISA for insulin (n = 10, 33, 9 and 24 mice) (c), proinsulin (n = 31, 44, 9 and 26 mice) (d), C-peptide (n = 10, 33, 9 and 24 mice) (e) and proinsulin/insulin ratio (n = 10, 33, 5 and 24 mice) (f). (g,h) Blood glucose levels in 12-week-old B10^k.Rag1^−/−, B10^k.Rag1^−/−.insHEL, NOD^k.Rag1^−/− and (non-diabetic) NOD^k.Rag1^−/−.insHEL mice following a glucose tolerance test (n = 28, 47, 9 and 21 mice) (g) or an insulin tolerance test (n = 8, 17, 3 and 13 mice) (h). Data are shown as means ± s.e.m. *P < 0.05, **P < 0.001, ***P < 0.0001.

Figure 3

Qualitative rather than quantitative differences in the UPR on the B10 and NOD backgrounds. (a) Globa principal-components analysis (PCA) of RNA-seq data for islets (n = 3 mice/group). (b) Schematic of the number of significant (corrected P < 0.05) gene expression differences for pairs of sample conditions. (c) Number of significant gene set changes in gene set enrichment analysis (GSEA) using cutoffs of P < 0.05 and P < 0.001. (d) ERAI mice were crossed to B10^k.Rag1^−/−.insHEL and NOD^k.Rag1^−/−.insHEL mice, and islets were analyzed by flow cytometry. Histograms show Xbp1s (Venus) expression in insulin-expressing beta cells. Results are representative of three experiments. (e) PCA of the Xbp1 response gene set in islets (n = 3 mice/group). (f) Scatterplot of individual Xbp1 response genes, showing the average log2-transformed fold change in expression induced by insHEL on the B10 background versus the NOD background. The dashed line indicates equivalent regulation; outlier genes are annotated. (g) Dot plot of mass spectrometry expression ratios, showing on each axis the log₂-transformed ratio of expression in B10^k.Rag1^−/−.insHEL islets as compared to NOD^k.Rag1^−/−.insHEL islets in duplicate experiments. All reproducibly detected proteins are displayed in gray, with black dots indicating annotated UPR-related proteins. (h,i) Immunofluorescence of wild-type and insHEL transgenic islets with a polyclonal antibody to insulin, antibody to Manf and DAPI. Staining is representative of three experiments (h), with Manf quantification (n = 10 mice/group) (i). Scale bar, 100 μm. Data are shown as means ± s.e.m. *P < 0.05, ***P < 0.0001. NS, not significant.

Figure 4

Genetic control of NOD mouse susceptibility to transgene-induced diabetes. (a) Diabetes incidence in insHEL transgenic male mice on the NOD^k background (n = 43) or on NOD^k congenic backgrounds homozygous for B6- or B10-derived chromosomal segments containing the diabetes resistance allele of Idd3 (n = 14), Idd5 (n = 10), Idd9 (n = 13), Idd3 and Idd5 (n = 12), and Idd3, Idd10 and Idd18 (n = 21). (b) A cohort of (NOD × B10)F2.Rag1^-/-.insHEL male mice (n = 331) was assessed for diabetes incidence at 28 weeks of age and genotyped for 740 informative SNPs. Quantitative trait locus (QTL) association is shown across the genome. LOD, logarithm of odds. (c,d) LOD scores for chromosomes 13 (c) and 19 (d), indicating the LOD support intervals (LOD drop of 1) for the associated Tid1, Tid2 and Tid3 loci. (e–g) Diabetes development in the (NOD × B10)F2.Rag1^-/-.insHEL cohort when stratified by genotype at the linkage SNP (rs13481783) in the Tid1 locus (n = 98, 157 and 75 mice) (e), at the linkage SNP (gnf13.088.732) in the Tid2 locus (n = 100, 163 and 67 mice) (f) and at the linkage SNP (rs6237466) in the Tid3 locus (n = 68, 166 and 96 mice) (g). (h) Diabetes development in the (NOD × B10)F2.Rag1^-/-.insHEL cohort with the B10 homozygous, B10/NOD heterozygous (Het) and NOD homozygous genotypes at all Tid loci (n = 12, 54 and 14 mice).

Figure 5

Xrcc4 mutation drives enhanced susceptibility to senescence. (a) Three-dimensional structure of the Lig4 complex, determined using the human structure as scaffolding. Modified residues are highlighted in red, and the arrow and dotted lines indicate regions of major instability. The box highlights the regions shown in detail in b and c. (b,c) Molecular interactions calculated along the represented trajectory for the B10 (b) and NOD (c) alleles of Xrcc4. (d) Per-residue root-mean-square fluctuation (RMSF) in Xrcc4 between 70–100 ns of simulation. Regions with a different fluctuation profile are highlighted by the dashed lines and one arrow, corresponding to the regions indicated in a. The results are representative of four simulations. (e,f) Representative immunoblotting of mouse embryonic fibroblasts (MEFs) for Xrcc4, Xlf and Lig4 (e), with quantification (n = 5 technical replicates/group) (f). (g) Proportion of MEFs that remained positive for H2A.X (Ser139) phosphorylation (pH2A.X; indicative of DSBs) after etoposide exposure (n = 6 technical replicates/group). (h) Wild-type CHO cells and Xrcc4-deficient (KO) CHO cells reconstituted with the B10 or NOD Xrcc4 allele were exposed to etoposide, and unrepaired DNA damage was quantified (n = 6–8 technical replicates/group). (i,j) Immunofluorescence with a polyclonal antibody to insulin, antibody to phosphorylated H2A.X and DAPI on pancreata from the B10^k.Rag1^−/− and NOD^k.Rag1^−/− backgrounds. Quantification is shown for islet raw fluorescence in the channel for phosphorylated H2A.X (n = 19, 17, 20 and 20 sections) (i), with images of representative sections (j). Scale bar, 100 μm. (k) Diabetes incidence of (B10^k × DBA/2)F1 and (B10^k × DBA/2)F1.insHEL mice (n = 6 and 11 mice). (l,m) Immunofluorescence with a polyclonal antibody to insulin, antibody to phosphorylated H2A.X and DAPI on pancreata from the B10^k and (B10^k × DBA/2)F1 backgrounds. Quantification is shown of islet raw fluorescence in the channel for phosphorylated H2A.X (n = 19, 17, 16 and 20 sections) (l), with images of representative sections (m). Scale bar, 100 μm. Data are shown as means ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.0001; NS, not significant.

Figure 6

Reduced Glis3 expression results in enhanced susceptibility to apoptosis. (a) Islet expression of Glis3, based on RNA-seq analysis (n = 3 mice/group). (b) The percentage of apoptotic cells in the islets of wild-type and insHEL transgenic B10^k.Rag1^-/- and NOD^k.Rag1^-/- mice after culturing in the presence of low (5 mM) and high (25 mM) concentrations of glucose (n = 5–6 replicates/group). (c) The percentage of apoptotic cells in the islets of B10^k.Rag1^-/- and NOD^k.Rag1^-/- cells, with and without insHEL, after culturing in the presence of the geldanamycin analog 17-DMAG and a low (5 mM) glucose concentration (n = 6–18 replicates/group). (d–f) Representative sections (d), average observation of apoptosis in islets (e) and beta cell mass (f) after immunohistochemistry with a polyclonal antibody to insulin and antibody to activated caspase-3 (aCasp3) (n = 4–5 replicates/group). In d, the box highlights the magnified region. Arrows indicate example apoptotic cells. Scale bars, 100 μm. (g) Diabetes incidence in B10 (n = 6), B10.Glis3^+/- (n = 11), B10.insHEL (n = 12) and B10.Glis3^+/-.insHEL (n = 23) littermates. (h,i) Pancreas immunofluorescence with a polyclonal antibody to insulin, antibody to Manf and DAPI. Fluorescence in the channel for Manf was quantified in islets (n = 8, 8, 10 and 10 mice) (h), with representative staining shown (i). Scale bar, 100 μm. Data are shown as means ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.0001; NS, not significant.

Figure 7

Dietary change recapitulates the effect of the NOD genetic background on a resistant mouse strain. B10^k.Rag1^−/−.insHEL mice were maintained on a control diet or converted to a 10% fat diet at 6 weeks of age. (a,b) Islet expression (quantitative PCR) of Glis3 (a) and Manf (b), normalized to Rpl37a expression (n = 6 mice/group). (c,d) Pancreas immunofluorescence with a polyclonal antibody to insulin, antibody to Manf and DAPI, with wild-type B10^k.Rag1^−/− mice and B10^k.Rag1^−/−.insHEL mice on a control diet and B10^k.Rag1^−/−.insHEL mice on a 10% fat diet. Quantification is shown of islet raw fluorescence in the channel for Manf (n = 8, 10 and 10) (c), with images of representative sections (d). Scale bar, 100 μm. (e) Diabetes incidence of B10^k.Rag1^−/−.insHEL mice fed with a control diet (n = 17) or a 10% fat diet (n = 9). Data are shown as means ± s.e.m. **P < 0.01, ***P < 0.0001.

Figure 8

Molecular changes in the islets of patients with T2D mirror the processes altered in NOD mice. mRNA expression in human pancreatic islets from healthy individuals (n = 105) and those diagnosed with T2D (n = 14) was assessed through RNA-seq analysis. (a) Relationship between GLIS3 and MANF expression in healthy ndividuals (Spearman correlation P value = 0.043), individuals with T2D (Spearman correlation P value = 0.075) and all individuals (Spearman correlation P value = 0.028). (b-e) Expression of XRCC4 (b) LIG4 (c), H2AFX (d) and CDKN1A (e) in healthy islets as compared to i slets from patients withT2D (P values shown after multiple-testing correction). The median and interquartile range (IQR; box) are shown, with error bars indicating 1.5 times the IQR. Individual values are shown if beyond 1.5 times the IQR. (f) Relationship between H2AFX and LIG4 expression in human islets (Spearman correlation P value = 5 × 10^-9).