Insulin promoter DNA methylation correlates negatively with insulin gene expression and positively with HbA(1c) levels in human pancreatic islets

Abstract

Aims/hypothesis: Although recent studies propose that epigenetic factors influence insulin expression, the regulation of the insulin gene in type 2 diabetic islets is still not fully understood. Here, we examined DNA methylation of the insulin gene promoter in pancreatic islets from patients with type 2 diabetes and non-diabetic human donors and related it to insulin expression, HbA(1c) levels, BMI and age.

Methods: DNA methylation was analysed in 25 CpG sites of the insulin promoter and insulin mRNA expression was analysed using quantitative RT-PCR in pancreatic islets from nine donors with type 2 diabetes and 48 non-diabetic donors.

Results: Insulin mRNA expression (p = 0.002), insulin content (p = 0.004) and glucose-stimulated insulin secretion (p = 0.04) were reduced in pancreatic islets from patients with type 2 diabetes compared with non-diabetic donors. Moreover, four CpG sites located 234 bp, 180 and 102 bp upstream and 63 bp downstream of the transcription start site (CpG -234, -180, -102 and +63, respectively), showed increased DNA methylation in type 2 diabetic compared with non-diabetic islets (7.8%, p = 0.03; 7.1%, p = 0.02; 4.4%, p = 0.03 and 9.3%, p = 0.03, respectively). While insulin mRNA expression correlated negatively (p < 1 × 10(-6)), the level of HbA(1c) correlated positively (p ≤ 0.01) with the degree of DNA methylation for CpG -234, -180 and +63. Furthermore, DNA methylation for nine additional CpG sites correlated negatively with insulin mRNA expression (p ≤ 0.01). Also, exposure to hyperglycaemia for 72 h increased insulin promoter DNA methylation in clonal rat beta cells (p = 0.005).

Conclusions/interpretations: This study demonstrates that DNA methylation of the insulin promoter is increased in patients with type 2 diabetes and correlates negatively with insulin gene expression in human pancreatic islets.

Fig. 1

Insulin mRNA levels, insulin content and glucose-stimulated insulin secretion measured in human pancreatic islets. Pancreatic islets from patients with type 2 diabetes show decreased (a) relative insulin mRNA levels, (b) insulin content and (c) glucose-stimulated insulin secretion, compared with non-diabetic control donors. Results are expressed as mean ± SEM. *p < 0.05 vs control islets. Control, non-diabetic donors; GSIS, glucose-stimulated insulin secretion; T2D, donors with type 2 diabetes

Fig. 2

DNA methylation of the insulin promoter. (a) A schematic representation of 2,518 bp of the insulin gene region analysed for DNA methylation. Positions of CpG sites in relation to the TSS are indicated: black circles represent analysed CpG sites, grey circles are not analysed. Putative and previously known transcription factor binding sites that co-localise with a CpG site are indicated. DNA methylation levels in percentage are analysed in (b) human pancreatic islets from 48 controls (white bars) and nine type 2 diabetic patients (black bars) as well as in five blood samples (grey bars; one man and four women, 29–57 years) and (c) beta cells (white bars) and alpha cells (black bars) isolated from pancreatic islets of three human donors. Results are expressed as mean ± SEM, and have not been corrected for multiple testing. ^† p < 0.05 vs control islets, *p < 0.05 vs control islets. BACH2, basic leucine zipper transcription factor 2; COUP, chicken ovalbumin upstream promoter; HNF4, hepatocyte nuclear factor 4; PDX1, pancreatic and duodenal homeobox 1; ISL1, islet 1; EVX1/2, even-skipped homeobox 1/2; MARE, Maf recognition element; NR2C1/2, nuclear receptor subfamily 2, group C, member 1/2; PTF1, pancreas specific transcription factor, NEUROD1, neurogenic differentiation 1

Fig. 3

Impact of type 2 diabetes on DNA methylation of the insulin promoter. Pancreatic islets from nine patients with type 2 diabetes show elevated insulin promoter DNA methylation in (a) CpG site −234, (b) CpG site −180, (c) CpG site −102 and (d) CpG site +63, compared with 48 non-diabetic donors. Results are expressed as mean ± SEM and have not been corrected for multiple testing. *p < 0.05. Control, non-diabetic donors; T2D, donors with type 2 diabetes

Fig. 4

Correlations between the level of insulin mRNA expression and insulin promoter DNA methylation in human pancreatic islets. The relative insulin mRNA expression correlates negatively with insulin promoter DNA methylation at (a) CpG site −234 (ρ = −0.64, p < 1 × 10⁻⁶), (b) CpG site −180 (ρ = −0.66, p < 1 × 10⁻⁶) and (d) CpG site +63 (ρ = −0.72, p < 1 × 10⁻⁶), while borderline significance was established for (c) CpG site −102 (ρ = −0.25, p = 0.065)

Fig. 5

Impact of hyperglycaemia on insulin promoter DNA methylation at (a) CpG site −234, (b) CpG site −180, (c) CpG site −102 and (d) CpG site +63. The HbA_1c level correlates positively with insulin promoter DNA methylation at (a) CpG site −234 (ρ=0.36, p=0.01), (b) CpG site −180 (ρ=0.42, p=0.004) and (d) CpG site +63 (ρ=0.44, p=0.002), while borderline significance was established for (c) CpG site −102 (ρ=0.28, p=0.056) in human pancreatic islets. (e) Clonal rat beta cells cultured in 16.7 mmol/l glucose showed increased insulin promoter DNA methylation of CpG sites −1,057 and +58 and a significant increase was also seen for the average DNA methylation measured across the studied region compared with beta cells cultured in 11.1 mmol/l glucose. Results are expressed as mean ± SEM. *p < 0.05, **p < 0.01. Black bars, beta cells cultured in 16.7 mmol/l glucose; white bars, beta cells cultured in 11.1 mmol/l glucose