Paternal allelic mutation at the Kcnq1 locus reduces pancreatic β-cell mass by epigenetic modification of Cdkn1c

Abstract

Genetic factors are important determinants of the onset and progression of diabetes mellitus. Numerous susceptibility genes for type 2 diabetes, including potassium voltage-gated channel, KQT-like subfamily Q, member1 (KCNQ1), have been identified in humans by genome-wide analyses and other studies. Experiments with genetically modified mice have also implicated various genes in the pathogenesis of diabetes. However, the possible effects of the parent of origin for diabetes susceptibility alleles on disease onset have remained unclear. Here, we show that a mutation at the Kcnq1 locus reduces pancreatic β-cell mass in mice by epigenetic modulation only when it is inherited from the father. The noncoding RNA KCNQ1 overlapping transcript1 (Kcnq1ot1) is expressed from the Kcnq1 locus and regulates the expression of neighboring genes on the paternal allele. We found that disruption of Kcnq1 results in reduced Kcnq1ot1 expression as well as the increased expression of cyclin-dependent kinase inhibitor 1C (Cdkn1c), an imprinted gene that encodes a cell cycle inhibitor, only when the mutation is on the paternal allele. Furthermore, histone modification at the Cdkn1c promoter region in pancreatic islets was found to contribute to this phenomenon. Our observations suggest that the Kcnq1 genomic region directly regulates pancreatic β-cell mass and that genomic imprinting may be a determinant of the onset of diabetes mellitus.

Keywords: Kcnq1; imprinting; pancreatic β-cells.

Fig. 1.

Effect of Kcnq1 mutation on pancreatic β-cell mass and glucose tolerance. (A) Insulin secretion by pancreatic islets isolated from 12-wk-old WT or Kcnq1^−/− mice and incubated for 30 min with 2.8 or 16.8 mM glucose, 500 µM tolbutamide (TB), or 30 mM KCl. Data were normalized by islet insulin content. (B) Birth weight of WT and Kcnq1 heterozygous KO (MH or PH) mice. (C, Left and D, Left) Normalized β-cell mass for WT, MH, and PH mice at (C) birth and (D) 24 wk of age after maintenance on a high-fat diet after weaning. The β-cell mass was determined as the ratio of the area positive for insulin immunostaining (red) to the total pancreatic area in sections similar to those shown in C, Right and D, Right. The pancreatic sections were also stained with antibodies to glucagon (green). (Scale bars: 50 µm.) (E) Fraction of Ki67-positive cells in the insulin-positive cells observed in 24-wk-old WT, MH, and PH mice. (F) Body weight as well as blood glucose and plasma insulin concentrations in the fed state for WT, MH, and PH mice at various ages or 12 wk of age (plasma insulin). (G) Blood glucose and plasma insulin levels of 24-wk-old WT, MH, and PH mice during an OGTT. All quantitative data are means ± SEM for (A) 7, (B) 6–20, (C) 5–9, (D) 8–11, (E) 4–5, (F) 5–37, or (G) 7–9 mice of each group. *P < 0.05; **P < 0.01 for the indicated comparisons or vs. the corresponding value for WT or MH mice.

Fig. S1.

Perfusion analysis of glucose-induced insulin secretion in WT or Kcnq1^−/− mice. Data were normalized by insulin content of the pancreas. Quantitative data are means ± SEM for five mice of each group.

Fig. 2.

Analysis and effects of gene imprinting in allele-specific Kcnq1 heterozygous KO mice. (A) Relative abundance of Kcnq1ot1 RNA in islets isolated from WT, MH, or PH mice at 6 wk of age. (B) Relative expression of genes in the Kcnq1 region in islets isolated from mice at 6 wk of age. (C) Cell proliferation and (D) immunoblot analysis of proliferating cell nuclear antigen (PCNA) and β-actin as loading controls for INS-1 cells overexpressing Kcnq1, Phlda2, Slc22a18, or Cdkn1c or those infected with the corresponding empty retrovirus. All quantitative data are means ± SEM for (A) five or (B) three to four mice of each group or (D) six independent experiments. *P < 0.05; **P < 0.01 for the indicated comparisons or vs. the corresponding value for cells infected with the empty virus.

Fig. S2.

(A) Immunoblot analysis of KCNQ1 in islets from 12-wk-old mice of the indicated Kcnq1 genotypes. (B) Blood glucose and (C) plasma insulin concentrations of WT and Kcnq1^+/− mice in the fed state at the indicated ages during maintenance on a normal control diet (NCD) or a high-fat diet (HFD). (D) Blood glucose and plasma insulin levels during an OGTT for 12-wk-old WT or Kcnq1^+/− mice fed an HFD. All quantitative data are means ± SEM for (B and C) 10–30 or (D) 3–4 mice of each group.

Fig. 3.

Effect of paternal Kcnq1 mutation on the expression of Cdkn1c. (A) Immunoblot analysis of CDKN1C in islets isolated from 1-wk-old WT, MH, or PH mice. (B) Relative expression of Cdkn1c in islets from mice of three groups at 1 wk of age. (C) ChIP analysis of the Cdkn1c promoter with antibodies to trimethylated H3K27 (H3K27me3) or acetylated H3K9/K14 in islets isolated from 6-wk-old mice. (Left) Representative and (Center and Right) quantitative data are shown. (D and E) Expression of Cdkn1c at the (D) protein and (E) mRNA levels in islets from 24-wk-old mice fed a high-fat diet. (F, Left) Representative immunostaining of CDKN1C and staining of nuclei with DAPI in islets from 24-wk-old mice fed a high-fat diet. (Scale bars: 10 µm.) (F, Right) The fluorescence intensity for CDKN1C normalized by that of DAPI was also determined. (G) Relative expression of Kcnq1ot1 in organs of three groups of mice. (H) ChIP analysis of the Cdkn1c promoter with antibodies to H3K27me3 in liver, brain, and epididymal white adipose tissue isolated from 6-wk-old mice. (I) Relative expression of Cdkn1c in liver, brain, and adipose tissue from 6-wk-old mice. All quantitative data are means ± SEM for (B) three to five, (C) six, (E) three to five, (F) four to eight, or (G–I) five to six mice of each group. *P < 0.05; **P < 0.01.

Fig. 4.

Effects of allele-specific truncation of Kcnq1ot1 on pancreatic β-cell mass, Cdkn1c expression in islets, and glucose tolerance. (A) Relative expression of Kcnq1ot1 in islets isolated from 6-wk-old WT mice, mice with Kcnq1ot1 truncation on the maternal allele (TM mice), or TP mice. (B and C) Pancreatic β-cell mass at (B) birth and (C) 24 wk of age in mice of three groups determined as in Fig. 1. (Scale bars: 50 µm.) (D) Relative expression of Cdkn1c in islets from 24-wk-old mice. (E) Blood glucose and plasma insulin levels during an OGTT in 24-wk-old mice. All quantitative data are means ± SEM for (A) 3–5, (B) 6–13, (C) 6–10, (D) 3–5, and (E) 6–10 mice. *P < 0.05; **P < 0.01 for the indicated comparisons or vs. the corresponding value for WT or TM mice.