Background: Epigenetic factors regulate tissue-specific expression and X-chromosome inactivation. Previous studies have identified epigenetic differences between sexes in some human tissues. However, it is unclear whether epigenetic modifications contribute to sex-specific differences in insulin secretion and metabolism. Here, we investigate the impact of sex on the genome-wide DNA methylation pattern in human pancreatic islets from 53 males and 34 females, and relate the methylome to changes in expression and insulin secretion. Results: Glucose-stimulated insulin secretion is higher in female versus male islets. Genome-wide DNA methylation data in human islets clusters based on sex. While the chromosome-wide DNA methylation level on the X-chromosome is higher in female versus male islets, the autosomes do not display a global methylation difference between sexes. Methylation of 8,140 individual X-chromosome sites and 470 autosomal sites shows sex-specific differences in human islets. These include sites in/near AR, DUSP9, HNF4A, BCL11A and CDKN2B. 61 X-chromosome genes and 18 autosomal genes display sex-specific differences in both DNA methylation and expression. These include NKAP, SPESP1 and APLN, which exhibited lower expression in females. Functional analyses demonstrate that methylation of NKAP and SPESP1 promoters in vitro suppresses their transcriptional activity. Silencing of Nkap or Apln in clonal beta-cells results in increased insulin secretion. Differential methylation between sexes is associated with altered levels of microRNAs miR-660 and miR-532 and related target genes. Conclusions: Chromosome-wide and gene-specific sex differences in DNA methylation associate with altered expression and insulin secretion in human islets. Our data demonstrate that epigenetics contribute to sex-specific metabolic phenotypes.

Circulating free fatty acids are often elevated in patients with type 2 diabetes (T2D) and obese individuals. Chronic exposure to high levels of saturated fatty acids has detrimental effects on islet function and insulin secretion. Altered gene expression and epigenetics may contribute to T2D and obesity. However, there is limited information on whether fatty acids alter the genome-wide transcriptome profile in conjunction with DNA methylation patterns in human pancreatic islets. To dissect the molecular mechanisms linking lipotoxicity to impaired insulin secretion, we investigated the effects of a 48 h palmitate treatment in vitro on genome-wide mRNA expression and DNA methylation patterns in human pancreatic islets. Genome-wide mRNA expression was analyzed using Affymetrix GeneChip® Human Gene 1.0 ST whole transcript-based array (n = 13) and genome-wide DNA methylation was analyzed using Infinium HumanMethylation450K BeadChip (n = 13) in human pancreatic islets exposed to palmitate or control media for 48 h. A non-parametric paired Wilcoxon statistical test was used to analyze mRNA expression. Apoptosis was measured using Apo-ONE® Homogeneous Caspase-3/7 Assay (n = 4). While glucose-stimulated insulin secretion was decreased, there was no significant effect on apoptosis in human islets exposed to palmitate. We identified 1,860 differentially expressed genes in palmitate-treated human islets. These include candidate genes for T2D, such as TCF7L2, GLIS3, HNF1B and SLC30A8. Additionally, genes in glycolysis/gluconeogenesis, pyruvate metabolism, fatty acid metabolism, glutathione metabolism and one carbon pool by folate were differentially expressed in palmitate-treated human islets. Palmitate treatment altered the global DNA methylation level and DNA methylation levels of CpG island shelves and shores, 5′UTR, 3′UTR and gene body regions in human islets. Moreover, 290 genes with differential expression had a corresponding change in DNA methylation, for example, TCF7L2 and GLIS3. Importantly, out of the genes differentially expressed due to palmitate treatment in human islets, 67 were also associated with BMI and 37 were differentially expressed in islets from T2D patients. Our study demonstrates that palmitate treatment of human pancreatic islets gives rise to epigenetic modifications that together with altered gene expression may contribute to impaired insulin secretion and T2D.

Background: Insulin secretion is enhanced upon the binding of Glucagon-like peptide-1 (GLP-1) to its receptor (GLP1R) in pancreatic β cells. Although a reduced expression of GLP1R in pancreatic islets from type 2 diabetic patients and hyperglycaemic rats has been established, it is still unknown if this is caused by differential DNA methylation of GLP1R in pancreatic islets of type 2 diabetic patients. Methods: In this study, DNA methylation levels of 12 CpG sites close to the transcription start site of GLP1R were analysed in pancreatic islets from 55 non-diabetic and 10 type 2 diabetic human donors as well as in β and α cells isolated from human pancreatic islets. DNA methylation of GLP1R was related to GLP1R expression, HbA_1c levels and BMI. Moreover, mRNA expression of MECP2, DNMT1, DNMT3A and DNMT3B was analysed in pancreatic islets of the non-diabetic and type 2 diabetic donors. Results: One CpG unit, at position +199 and +205 bp from the transcription start site, showed a small increase in DNA methylation in islets from donors with type 2 diabetes compared to non-diabetic donors (0.53%, p=0.02). Furthermore, DNA methylation levels of one CpG site located 376 bp upstream of the transcription start site of GLP1R correlated negatively with GLP1R expression (rho=−0.34, p=0.008) but positively with BMI and HbA_1c (rho=0.30, p=0.02 and rho=0.30, p=0.03, respectively). This specific CpG site is located in an area with known SP1 and SP3 transcription factor binding sites. Moreover, when we compared the DNA methylation of the GLP1R promoter in isolated human β and α cells, we found that it was higher in α- compared with β-cells (p=0.009). Finally, there was a trend towards decreased DNMT3A expression (p=0.056) in type 2 diabetic compared with non-diabetic islets. Conclusions: Together, our study shows that while BMI and HbA_1c are positively associated with DNA methylation levels of GLP1R, its expression is negatively associated with DNA methylation of GLP1R in human pancreatic islets.