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. 2016 Mar 31:7:11089.
doi: 10.1038/ncomms11089.

Blood-based biomarkers of age-associated epigenetic changes in human islets associate with insulin secretion and diabetes

Karl Bacos  1 , Linn Gillberg  2   3 , Petr Volkov  1 , Anders H Olsson  2 , Torben Hansen  4 , Oluf Pedersen  4 , Anette Prior Gjesing  4 , Hans Eiberg  5 , Tiinamaija Tuomi  6   7 , Peter Almgren  8 , Leif Groop  7   8 , Lena Eliasson  9 , Allan Vaag  2   3 , Tasnim Dayeh  1 , Charlotte Ling  1
Affiliations

Blood-based biomarkers of age-associated epigenetic changes in human islets associate with insulin secretion and diabetes

Karl Bacos et al. Nat Commun. .

Abstract

Aging associates with impaired pancreatic islet function and increased type 2 diabetes (T2D) risk. Here we examine whether age-related epigenetic changes affect human islet function and if blood-based epigenetic biomarkers reflect these changes and associate with future T2D. We analyse DNA methylation genome-wide in islets from 87 non-diabetic donors, aged 26-74 years. Aging associates with increased DNA methylation of 241 sites. These sites cover loci previously associated with T2D, for example, KLF14. Blood-based epigenetic biomarkers reflect age-related methylation changes in 83 genes identified in human islets (for example, KLF14, FHL2, ZNF518B and FAM123C) and some associate with insulin secretion and T2D. DNA methylation correlates with islet expression of multiple genes, including FHL2, ZNF518B, GNPNAT1 and HLTF. Silencing these genes in β-cells alter insulin secretion. Together, we demonstrate that blood-based epigenetic biomarkers reflect age-related DNA methylation changes in human islets, and associate with insulin secretion in vivo and T2D.

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Figures

Figure 1
Figure 1. Aging is associated with altered DNA methylation in human pancreatic islets.
(a) Correlation between age and methylation of four CpG sites with the most significant associations with age in pancreatic islets as analysed by linear regression analysis (n=87). (b) Distribution of the CpG sites significantly associated with age in relation to gene regions. TSS1500, region 200–1,500 bp upstream of the transcription start site (TSS). TSS200, 200 bp immediately upstream of the TSS. UTR, untranslated region. (c) Distribution of the CpG sites significantly associated with age in relation to CpG islands. Northern/Southern shore: 2 kb regions upstream/downstream of the CpG island. Northern/Southern shelf: 2 kb regions outside the shore regions. Open sea: all DNA outside the CpG island regions. *P<0.05, **P<0.01 and ***P<0.001. Significance of the data in b and c were calculated with a χ2-test and have been corrected with Bonferroni correction.
Figure 2
Figure 2. Age-associated methylation changes of genes with importance for diabetes, β-cell function and mitochondria.
(a) Graphic illustration of the results of a literature study performed on all genes with age-associated methylation changes in human islets. PubMed was searched for papers containing the gene name and each of the following search terms: diabetes, pancreatic islet, pancreatic β-cell or mitochondrial function. Of note, a few genes are included in more than one of the groups. (b–g) Correlation between age and DNA methylation of CpG sites in genes associated with diabetes risk; CCND2 (b), CILP2 (c), PBX4 (d), SH2B3 (e), SLC6A4 (f) and TCF7 (g) as analysed with linear regression analysis (n=87).
Figure 3
Figure 3. Correlations between DNA methylation and gene expression in human pancreatic islets.
DNA methylation of CpG sites in FHL2 (a), ZNF518B (b), GNPNAT1 (c), and HLTF (d) and the expression of respective gene correlates significantly in human pancreatic islets (Spearman correlation, n=87). q values were calculated with the standard Benjamini–Hochberg procedure. (e) ZNF518B and GNPNAT1 promoters were tested for their transcriptional activity in luciferase assays after mock-methylation or methylation with HhaI (methylates the internal CpG site in GCGC sequences) or SssI (methylates all CpG sites). Data are presented as mean±s.e.m. of six experiments with three biological replicates in each. *P<0.05 compared to control as analysed by a Wilcoxon signed rank test.
Figure 4
Figure 4. Knockdown of Fhl2, Zfp518b, Gnpnat1 and Hltf affects insulin secretion in clonal β-cells.
(a) Quantification of siRNA-mediated knockdown of Fhl2, Zfp518b, Gnpnat1 and Hltf in clonal β-cells. Data are presented as the mean±s.e.m. of seven experiments and were analysed with a Mann–Whitney test. ***P<0.001 versus negative control siRNA (siNC). (b) Insulin secretion at basal (2.8 mM) and/or stimulatory (16.7 mM) glucose levels were altered in β-cells deficient for Fhl2, Zfp518b, Gnpnat1 or Hltf. Data are presented as the mean±s.e.m. of seven experiments with three biological replicates in each and were analysed with a Wilcoxon signed rank test. *P<0.05 versus siNC 16.7 mM and #P<0.05 versus siNC 2.8 mM. (c) The fold change of insulin secretion (the ratio of secretion at 16.7 mM and 2.8 mM glucose) was reduced in β-cells deficient for Fhl2, while it was increased in cells deficient for Zfp518b, Gnpnat1 or Hltf. Data are presented as mean±s.e.m. of seven experiments and were analysed with a Mann–Whitney test. *P<0.05 versus siNC and **P<0.01 versus siNC.
Figure 5
Figure 5. Methylation changes of KLF14, FHL2, FAM123C and ZNF518B in human islets are reflected by methylation changes in blood.
Association between age and DNA methylation of sites in KLF14 (a), FHL2 (b), FAM123C (c) and ZNF518B (d) in pancreatic islets from the human islet cohort (n=87, upper panels) and blood from the Danish Family Study at baseline (n=112, lower panels), as analysed by linear regression analysis.
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