Increased DNA methylation and decreased expression of PDX-1 in pancreatic islets from patients with type 2 diabetes

Abstract

Mutations in pancreatic duodenal homeobox 1 (PDX-1) can cause a monogenic form of diabetes (maturity onset diabetes of the young 4) in humans, and silencing Pdx-1 in pancreatic β-cells of mice causes diabetes. However, it is not established whether epigenetic alterations of PDX-1 influence type 2 diabetes (T2D) in humans. Here we analyzed mRNA expression and DNA methylation of PDX-1 in human pancreatic islets from 55 nondiabetic donors and nine patients with T2D. We further studied epigenetic regulation of PDX-1 in clonal β-cells. PDX-1 expression was decreased in pancreatic islets from patients with T2D compared with nondiabetic donors (P = 0.0002) and correlated positively with insulin expression (rho = 0.59, P = 0.000001) and glucose-stimulated insulin secretion (rho = 0.41, P = 0.005) in the human islets. Ten CpG sites in the distal PDX-1 promoter and enhancer regions exhibited significantly increased DNA methylation in islets from patients with T2D compared with nondiabetic donors. DNA methylation of PDX-1 correlated negatively with its gene expression in the human islets (rho = -0.64, P = 0.0000029). Moreover, methylation of the human PDX-1 promoter and enhancer regions suppressed reporter gene expression in clonal β-cells (P = 0.04). Our data further indicate that hyperglycemia decreases gene expression and increases DNA methylation of PDX-1 because glycosylated hemoglobin (HbA1c) correlates negatively with mRNA expression (rho = -0.50, P = 0.0004) and positively with DNA methylation (rho = 0.54, P = 0.00024) of PDX-1 in the human islets. Furthermore, while Pdx-1 expression decreased, Pdx-1 methylation and Dnmt1 expression increased in clonal β-cells exposed to high glucose. Overall, epigenetic modifications of PDX-1 may play a role in the development of T2D, given that pancreatic islets from patients with T2D and β-cells exposed to hyperglycemia exhibited increased DNA methylation and decreased expression of PDX-1. The expression levels of PDX-1 were further associated with insulin secretion in the human islets.

Fig. 1.

PDX-1 mRNA expression levels in human pancreatic islets from nondiabetic donors and patients with T2D. Expression was analyzed using quantitative RT-PCR. Results are expressed as mean ± sem. *, P < 0.05, nondiabetic vs. T2D islets.

Fig. 2.

Impact of T2D on DNA methylation of PDX-1 in human pancreatic islets. A, A schematic representation of 3500 bp of the human PDX-1 promoter and enhancer region. The three regions analyzed for DNA methylation, representing 93 bp of the proximal promoter, 436 bp of the distal promoter, and 475 bp of the enhancer region, are visualized. DNA methylation of the proximal PDX-1 promoter (B), distal PDX-1 promoter (C), and enhancer region (D) in human pancreatic islets of nondiabetic donors (white bars) and patients with T2D (black bars). Results are expressed as mean ± sem. FDR was used to correct for multiple testing with *, Q < 0.05 nondiabetic vs. T2D islets.

Fig. 3.

Impact of DNA methylation on gene expression of the human PDX-1 gene. A, An inverse correlation between PDX-1 mRNA expression and DNA methylation of CpG site −3534 in human pancreatic islets. B, A diagram of the three luciferase reporter plasmids used to test the effect of DNA methylation on PDX-1 promoter activity and the empty vector is visualized. The three plasmids contain either 908 bp of the human PDX-1 promoter, 606 bp of the human PDX-1 enhancer region, or 3800 bp of a sequence containing both the PDX-1 promoter and the enhancer region inserted into a pCpGL-basic vector. Methylated (gray and black bars) or mock-methylated (white bars) PDX-1 promoter and/or the enhancer constructs were transfected into clonal rat β-cells for 48 h before luciferase assay. The data were normalized with cotransfected renilla luciferase control vector and are the average from four separate experiments of five replicates each. Data are presented as relative expression compared with the nonmethylated construct including both the enhancer and promoter region. Results are expressed as mean ± sem. *, P < 0.05.

Fig. 4.

Impact of hyperglycemia on DNA methylation of PDX-1 and gene expression of Pdx-1 and Dnmt1. A, The HbA1c level correlated positively with DNA methylation of CpG site −3534 in human pancreatic islets. Clonal rat β-cells cultured in 16.7 mm glucose (black bars) for 72 h exhibit (B) decreased Pdx-1 mRNA expression and (C) nominally increased DNA methylation of two CpG sites of the Pdx-1 promoter compared with β-cells cultured in 11.1 mm (white bars). Results are expressed as mean ± sem and represent six independent experiments. D, High (16.7 mm) compared with low (2.7 mm) glucose for 48 h increased the mRNA expression of Dnmt1 but not Dnmt3a or Dnmt3b in clonal rat β-cells (n = 4). Results are expressed as mean ± sem. *, P < 0.05.

Fig. 5.

DNA methylation of PDX-1 in α-cells (white bars) and β-cells (black bars). Cells were isolated from pancreatic islets of three human donors. Results are expressed as mean ± sem.