High-Fat Diet-Fed Kcnq1 Mutant Mice Have Reduced Pancreatic β-Cell Mass via Gene-Environment Interaction

Article information

Diabetes Metab J. 2026;50(1):77-89

Publication date (electronic) : 2025 July 30

doi : https://doi.org/10.4093/dmj.2024.0790

¹Division of Diabetes and Endocrinology, Department of Internal Medicine, Kobe University Graduate School of Medicine, Kobe, Japan

²Division of Medical Chemistry, Department of Metabolism and Diseases, Kobe University Graduate School of Health Sciences, Kobe, Japan

³Metabolism and Nutrition Research Unit, Institute for Frontier Science Initiative, Kanazawa University, Kanazawa, Japan

⁴Laboratory for Animal Resources and Genetic Engineering, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan

⁵Department of Molecular Metabolic Regulation, Diabetes Research Center, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan

⁶Anticancer Strategies Laboratory, Advanced Research Initiative, Institute of Science Tokyo, Tokyo, Japan

⁷The Institute of Medical Science, Asahi Life Foundation, Tokyo, Japan

Corresponding author: Shun-ichiro Asahara https://orcid.org/0000-0001-6661-9402 Division of Diabetes and Endocrinology, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017, Japan E-mail: asahara@med.kobe-u.ac.jp

Received 2024 December 5; Accepted 2024 December 30.

Abstract

Background

The potassium voltage-gated channel subfamily Q member 1 (KCNQ1) gene has recently received much attention as a candidate susceptibility gene for type 2 diabetes mellitus, especially in Asian populations. We previously reported that Kcnq1 mutant mice exhibit reduced insulin secretion and hyperglycemia due to a decrease in pancreatic β-cell mass. Through in vivo and in vitro analyses, we ascertained that this mechanism is the result of the downregulation of the non-coding RNA ‘Kcnq1ot1,’ which is expressed in the paternal allele of the Kcnq1 gene region, causing an increase in the expression of the cell cycle inhibitor cyclin dependent kinase inhibitor 1C (Cdkn1c). It was found that decreased Kcnq1ot1 expression resulted in pancreatic β-cell failure; however, the degree of pancreatic β-cell volume reduction was not severe.

Methods

We induced obesity in Kcnq1ot1 truncation mice by feeding them a high-fat diet and evaluated pancreatic β-cell mass.

Results

In the present study, we reveal that CCAAT/enhancer binding protein beta (C/EBPβ), which is expressed at higher levels in pancreatic β-cells in obese individuals, further increases the expression of Cdkn1c, which is upregulated by the Kcnq1 gene mutation. We found that simultaneous Cdkn1c hypomethylation and C/EBPβ overexpression in pancreatic β-cells causes a synergistic decrease in pancreatic β-cell mass.

Conclusion

This finding suggests that the synergistic effect of genetic factors such as Kcnq1 gene mutations and environmental factors such as obesity and overeating, which lead to increased expression of C/EBPβ, contribute to the regulation of pancreatic β-cell mass. This study is the first to show that the Kcnq1 gene is related to pancreatic β-cell mass through genetic-environment interactions.

Keywords: CCAAT-enhancer-binding protein-beta; Cyclin-dependent kinase inhibitor p57; Diabetes mellitus; Gene-environment interaction; Insulin-secreting cells; KCNQ1OT1 RNA

GRAPHICAL ABSTRACT

Highlights

• Reduced Kcnq1ot1 expression in the islets led to changes in epigenetic regulation.

• Cdkn1c was upregulated with C/EBPβ expression and epigenetic changes in β-cells.

• HFD-fed Kcnq1ot1-reduced mice showed decreased β-cell mass.

• Cdkn1c deficiency restored β-cell mass in HFD-fed Kcnq1ot1-reduced mice.

INTRODUCTION

The global population of patients with type 2 diabetes mellitus (T2DM) continues to grow at a rapid pace, presenting a serious challenge. In 2008, a genome-wide association study (GWAS) of East Asians found that single nucleotide polymorphisms (SNPs) in intron 15 of the potassium voltage-gated channel subfamily Q member 1 (KCNQ1) gene on chromosome 11 were significant risk factors for T2DM [1,2]. Subsequent reports from institutions around the world have shown that populations with SNPs in the KCNQ1 gene exhibit decreased insulin secretion [3,4]. KCNQ1 is involved in the construction of voltage-gated channels and may also be involved in repolarization in cardiomyocytes [5]. It is known that KCNQ1 and its neighboring genes are imprinted genes whose expression is regulated by a long non-coding RNA ‘KCNQ1 opposite strand/antisense transcript 1 (KCNQ1OT1)’ expressed within the KCNQ1 gene region, and that KCNQ1 is expressed only in the maternal allele [6].

In a previous mouse study, we found that a mutation in the paternal allele of the Kcnq1 gene region decreased the expression of Kcnq1ot1 in pancreatic islets and increased the expression of one of its neighboring genes, cyclin dependent kinase inhibitor 1C (CDKN1C) [7]. Also known as p57, CDKN1C is a cell cycle inhibitor, and its upregulation caused a decrease in pancreatic β-cell volume and impaired glucose tolerance [8].

It remains unclear whether there is a correlation between the Kcnq1 mutations and SNPs we used, but it is possible that the onset of T2DM is related to changes in the expression levels of Kcnq1ot1 and Cdkn1c. However, in the previous paper, the decrease in pancreatic β-cell volume in Kcnq1 mutant mice was about 30%, which did not affect normal blood glucose levels [7]. In other words, the effect of Cdkn1c expression in pancreatic β-cells on glucose tolerance remains poorly understood. Recently, gene-environment interactions have been attracting increasing attention. Epigenetics is a representative phenomenon, and it has been reported that the prenatal environment and other factors can alter gene expression [9]. In adults, it has been reported that obesity and diet alter gene expression and molecular activity [10,11]. However, there are few reports on the association between disease susceptibility genes and environmental factors. Since World War II, the “westernization of food” has led to changes in the types and amounts of nutrients consumed in East Asia. Although caloric intake has not changed substantially, a large increase in lipid intake has been reported [12,13]. The recent explosion in the population of people with T2DM in East Asia may be due in large part to this increased intake of lipids.

In this study, we focused on Kcnq1ot1, which regulates the expression of the Kcnq1 and its neighboring genes, and examined the effects of a high-fat diet (HFD) on pancreatic β-cell mass and glucose tolerance using Kcnq1ot1 truncation mice, in which Kcnq1ot1 expression is decreased. The results revealed a significant effect on pancreatic β-cell mass and glucose tolerance. In addition, the transcription factor CCAAT/enhancer binding protein beta (C/EBPβ), which accumulated in islets because of the HFD, was found to bind to the CDKN1C promoter with a loose chromatin structure, resulting in increased expression of p57 and a marked reduction in pancreatic β-cell volume. It was shown that the simultaneous downregulation of Kcnq1ot1 expression (a genetic factor), and accumulation of C/EBPβ (an environmental factor), synergistically reduced pancreatic β-cell volume. This mechanism of pancreatic β-cell insufficiency may have contributed to the recent explosion in the population of East Asian patients with T2DM.

METHODS

Mice

Kcnq1ot1 truncation mice and β-cell–specific p57 knockout (KO) mice were generated as previously reported [7,14-16]. JF1 mice were provided by Dr. Shiroishi of the National Institute of Genetics. Pancreatic β-cell–specific HA-tagged C/EBPβ transgenic (TG) mice (Accession No. CDB0550T: https://large.riken.jp/distribution/mutant-list.html) were generated as follows. A pBlueScript vector containing the promoter of rat insulin 2 (Ins2) was provided by P.L. Herrera (University of Geneva Medical School, Geneva, Switzerland). We isolated a 3×HAtag and C/EBPβ cDNA by polymerase chain reaction (PCR) amplification and inserted it into the EcoRI and ClaI sites of the vector downstream of the insulin gene promoter. The resulting construct was linearized and microinjected into pronuclear zygotes of C57BL/6N mice. The resulting offspring were screened for transgene transmission by PCR analysis, with forward primer (5´-CTGGTCATCATCCTGCCTTT-3´), reverse primer (5´-CACTTCCATG-GGTCTAAAGG-3´). The PCR amplicon size was approximately 200 bp. The study protocol, which conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines, was approved by the Institutional Animal Care and Use Committee of RIKEN Kobe Branch and the Animal Ethics Committee of Kobe University Graduate School of Medicine (Approval No. P200905). For complete details regarding the mice, please see the Supplementary Methods.

Analytical procedures

Chromatin immunoprecipitation (ChIP) analysis was performed as previously described [17]. Tail vein blood samples were obtained to determine blood glucose and plasma insulin concentrations in the mice. RNA extracted from islets pooled from three to five mice was subjected to reverse transcription and real-time PCR analysis as previously described [18]. Lysates of isolated islets were prepared as described previously [19] and probed with antibodies to CDKN1C (Santa Cruz Biotechnology, Dallas, TX, USA), C/EBPβ (Santa Cruz Biotechnology), HA (Roche, Basel, Switzerland), and β-actin (Sigma-Aldrich, St. Louis, MO, USA). After sectioning of the pancreas, quantitation of β-cell mass was performed as previously described [20]. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed to detect proliferating cells. Then, proliferation and apoptosis in pancreatic β-cells were determined.

To confirm that C/EBPβ binds to the Cdkn1c promoter in vivo, MIN6 cells were assayed for luciferase activity at 48 hours after transfection, as previously reported [15]. To examine DNA methylation in the Cdkn1c promoter region of truncated paternal (TP) mice and wild-type (WT) mice, the quality and quantity of genomic DNA was assessed as previously described [17]. MIN6 cells were cultured, and an analysis of epigenetic control and post-translational modifications was performed as previously described [7,17]. For complete details of the analytical procedures, please see the Supplementary Methods.

Statistical analysis

Data are presented as mean±standard error of the mean. Comparisons were performed using analysis of variance followed by a two-tailed Student’s t-test. Differences were considered to be statistically significant when P<0.05.

RESULTS

Interaction between Cdkn1c promoter demethylation and increased C/EBPβ expression in pancreatic β-cells

We previously investigated pancreatic β-cell mass and glucose tolerance in mice with polyA-truncated Kcnq1ot1 [7]. The results showed that Kcnq1ot1 expression in pancreatic islets was decreased in TP mice with polyA inherited from the father compared with truncated maternal (TM) mice with polyA inherited from the mother as well as WT mice [14], and that they also showed decreased pancreatic β-cell volume and impaired glucose tolerance [7]. TP mice showed increased Cdkn1c expression in islets, which was thought to be due to a disruption in imprinting caused by decreased Kcnq1ot1 expression. To confirm the occurrence of imprinting dysregulation in the islets of TP mice with truncated Kcnq1ot1 in the paternal allele, we mated male C57B6 strain Kcnq1ot1 truncation mice with female JF1 strain WT mice. Then, we extracted RNA from islets isolated from the litters and performed cDNA sequencing analysis of Cdkn1c (Fig. 1A). The results showed that Cdkn1c cDNA was reverse-transcribed from the isolated islet RNA of TM mice and that WT mice expressed maternal JF1 lineage-derived cDNA, which is originally expressed by imprinting (Fig. 1B). Meanwhile, in the Cdkn1c cDNA from the isolated islet RNA of TP mice, the sequencing results revealed that both paternal B6-derived and maternal JF1-derived cDNAs were expressed. In other words, imprinting regulation is disrupted in the islets of TP mice, and Cdkn1c is expressed in the paternal allele, which does not normally express Cdkn1c (Fig. 1B). Next, to analyze epigenomic regulation in the islets of Kcnq1ot1 truncation mice, we analyzed histone modifications. Kcnq1ot1 recruits EZH2, a histone methyltransferase responsible for H3K27 trimethylation, to the promoter region of the Kcnq1 locus [21]. In other words, Kcnq1ot1 functions to repress the expression of the paternal allele gene cluster via H3K27 trimethylation of the promoter in the Kcnq1 gene region. Therefore, we used ChIP assay to examine H3K27 trimethylation at the Cdkn1c promoter using islets of TP, TM, and WT mice. We found that H3K27 trimethylation at the Cdkn1c promoter was decreased in islets from TP mice compared with TM and WT mice (Fig. 1C). Similarly, H3K9/14 acetylation, a marker of transcription promotion, was confirmed by ChIP assay, and significantly increased acetylation was observed in TP (Fig. 1C). These results indicate that Cdkn1c expression is upregulated in the islets of TP mice via histone modification. Furthermore, because Kcnq1ot1 has been reported to contribute to the recruitment of DNA methyltransferase (DNMT) [22], we used bisulfite sequence analysis to examine DNA methylation in the Cdkn1c promoter region of TP mice and WT mice. In the islets of WT mice, DNA methylation of the Cdkn1c promoter is thought to differ between paternal and maternal alleles. In fact, approximately 40% to 50% of cytosine-phosphate-guanine (CpG) regions in the islets of WT mice are methylated; these methylated CpG regions are considered paternal alleles, while the unmethylated CpG regions are considered maternal alleles (Fig. 1D). In contrast, the Cdkn1c promoter in the islets of TP mice has an overall unmethylated CpG region, suggesting that loss of Kcnq1ot1 function affects DNA methylation in the paternal allele (Fig. 1D).

Fig. 1.

Imprinting regulation in pancreatic islets of KCNQ1 opposite strand/antisense transcript 1 (Kcnq1ot1) truncation mice. (A) Schema of detection of cyclin dependent kinase inhibitor 1C (Cdkn1c) imprinting in pancreatic islets. Embryos of Kcnq1ot1 truncated paternal mice (TP mice) from wild-type (WT) JF1 female and Kcnq1ot1 truncation B6 male parents have single nucleotide polymorphisms derived from their parents. (B) Sequence of Cdkn1c mRNA from pancreatic islets of WT mice and TP mice. (C) Chromatin immunoprecipitation (ChIP) analysis of Cdkn1c promoter with antibodies to trimethylated H3K27 (H3K27me3) or acetylated H3K9/14 in islets isolated from each mouse. (D) Bisulfite sequence of the Cdkn1c promoter from pancreatic islets isolated from each mouse. Black and white circles indicated methylated and unmethylated CpG, respectively. All quantitative data are mean±standard error of the mean for (C) 3–5 mice in each group. RT-PCR, reverse transcription polymerase chain reaction; TM, truncated maternal. ^aP<0.01.

Cdkn1c is a cell cycle regulator and is thought to contribute to cell growth inhibition and apoptosis enhancement. It has been reported to play an important role in the regulation of pancreatic β-cell volume, and disruption of imprinting regulation by KCNQ1 gene mutations might be an important initiator in the regulation of Cdkn1c expression. Sp1 is known to be a transcription factor for Cdkn1c expression [23], and the promoter region also contains a transcription factor the C/EBP family binding motif, the CCAAT sequence [24]. It is not known whether the C/EBP family increases Cdkn1c expression in pancreatic β-cells. The interaction between the Cdkn1c promoter and C/EBPβ was confirmed in the mouse pancreatic β-cell line MIN6 cells by luciferase assays, which showed that C/EBPβ overexpression enhanced luciferase activity (Fig. 2A). However, overexpression of C/EBPβ in MIN6 cells did not significantly alter Cdkn1c expression compared with controls (Fig. 2B). Kcnq1ot1 recruits histone deacetylases (HDACs), histone methyltransferases, and DNMTs to each promoter in the Kcnq1 gene region [21,22]. Thus, Cdkn1c expression was confirmed by simultaneously administering the following inhibitors of epigenome-modifying enzymes: HDAC inhibitors, histone methyltransferase inhibitors, and DNMT inhibitors. The results revealed that Cdkn1c expression was enhanced by administration of the inhibitor cocktail and was further enhanced by C/EBPβ overexpression (Fig. 2B). Next, we examined whether the inhibitor cocktail affected the binding of C/EBPβ to the Cdkn1c promoter. The ChIP assay indicated that the binding of C/EBPβ to the Cdkn1c promoter region was enhanced by the inhibitor cocktail (Fig. 2C). In other words, DNA demethylation and open chromatinization enhance transcription of Cdkn1c by the transcription factor C/EBPβ.

Fig. 2.

Interaction of cyclin dependent kinase inhibitor 1C (Cdkn1c) expression between CCAAT/enhancer binding protein beta (C/EBPβ) and epigenetic regulation in pancreatic β-cell line. (A) Luciferase assays were performed to examine the effect of C/EBPβ on the transcription activation of Cdkn1c in MIN6 cells. (B) Quantitative reverse transcription polymerase chain reaction analysis of the effect of C/EBPβ with or without inhibitor cocktail (trichostatin A, 5-azacitidine, 3-deazaneplanocin A [Dznep]) on Cdkn1c expression in MIN6 cells. (C) Chromatin immunoprecipitation (ChIP) analysis of the binding of C/EBPβ to the Cdkn1c promoter in MIN6 cells. (Left) Representative and (right) quantitative data are shown. All quantitative data are mean±standard error of the mean for (A) 6, (B) 6–8, or (C) 5 mice in each group. HA, hemagglutin; IP, immunoprecipitation. ^aP<0.05, ^bP<0.01.

Effect of decreased Kcnq1ot1 expression and increased C/EBPβ expression on pancreatic β-cell volume

In vitro studies have shown that simultaneous epigenetic dysregulation and upregulation of C/EBPβ expression leads to upregulation of Cdkn1c expression in pancreatic β-cells. Next, we tested whether the same phenomenon occurs in vivo by breeding TG mice. Kcnq1ot1 truncated C/EBPβ-overexpressing (KT) mice were generated, and littermate TP mice, TG mice, and WT mice were used as controls. Blood glucose levels were measured randomly, and TG mice showed higher blood glucose levels compared with TP and WT mice. However, KT mice showed an even more pronounced hyperglycemia of >500 mg/dL despite their very light body weight (Fig. 3A and B). Next, serum insulin levels were measured at the age of 8 weeks. Consistent with the blood glucose data, TG mice showed lower serum insulin levels compared with WT and TP, while KT showed more markedly lower insulin levels (Fig. 3C). Measurements of pancreatic β-cell area and mass showed a 40% decrease in TP and TG compared with WT but an approximately 85% to 90% decrease in KT compared with WT, which may have been the cause of the decrease in insulin secretion (Fig. 3D and E). The in vitro analysis revealed that epigenetics modification changes mimicking decreased Kcnq1ot1 expression increased Cdkn1c expression. Meanwhile, the in vivo analysis revealed that Cdkn1c expression was upregulated in KT at both the protein and mRNA levels (Fig. 3F and G). To elucidate the mechanism of the decrease in pancreatic β-cell volume in KT, we evaluated the proliferative capacity and occurrence of apoptosis in pancreatic β-cells. The results showed that Ki67-positive pancreatic β-cells were significantly decreased and TUNEL-positive pancreatic β-cells were increased in KT compared with the control group (Fig. 3H and I).

Fig. 3.

Generation and analysis of KCNQ1 opposite strand/antisense transcript 1 (Kcnq1ot1) truncated CCAAT/enhancer binding protein beta (C/EBPβ)-overexpressing mice. (A) Blood glucose levels in the fed state for wild-type (WT), truncated paternal (TP), transgenic (TG), and Kcnq1ot1 truncated C/EBPβ-overexpressing (KT) mice at various ages. (B) Body weight for each mouse at the age of 10 weeks. (C) Serum insulin concentrations in the fed state with normal chow diet (NCD) for each mouse at the age of 8 weeks. (D) Immunostaining of insulin (red) and glucagon (green) in pancreatic sections from each 10-week-old NCD-fed mouse. (Left) Quantitative and (Right) representative data are shown (scale bars: 50 μm). (E) Quantification of pancreatic β-cell mass in each 10-week-old mouse. (F) Immunoblot analysis of cyclin dependent kinase inhibitor 1C (CDKN1C) and C/EBPβ in islets isolated from each 10-week-old mouse. (G) Relative expression of Cdkn1c mRNA in islets isolated from each 10-week-old mouse. (H) Percentage of Ki67+ β-cells per islet in each 10-week-old mouse. (I) The percentage of apoptotic β-cells per islet in each mouse was determined using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. (J) Construct of pancreatic β-cell–specific hemagglutin (HA)-tagged C/EBPβ transgenic mice. The arrows indicate sense and antisense primers. (K) Immunoblot analysis of HA in islets isolated from HA-tagged C/EBPβ transgenic mice. (L) Chromatin immunoprecipitation (ChIP) analysis of the Cdkn1c promoter with antibodies to HA-tag in islets isolated from each mouse. Representative (upper) and quantitative (lower) data are shown. All quantitative data are mean±standard error of the mean for (A) 5–8, (B) 10, (C) 7–8, (D, E) 5–8, (G) 5–6, (H) 7–8, (I) 5–7, or (L) 5 mice in each group. ^aP<0.05, ^bP<0.01.

Next, to confirm that C/EBPβ binds to the Cdkn1c promoter in vivo, we decided to perform a ChIP assay using isolated islets from KT mice. However, because we could not find a suitable C/EBPβ antibody to perform the ChIP assay, we generated mice overexpressing HA-tagged C/EBPβ in pancreatic β-cells (Fig. 3J and K) and bred them with Kcnq1ot1 truncated mice. Islets were isolated from the offspring of those mice, which we called Kcnq1ot1 truncated HA-C/EBPβ-overexpressing (HA-KT) mice, and ChIP assay was performed using extracted chromatin DNA. The results indicated that, similar to the in vitro analysis, C/EBPβ and Cdkn1c promoter were significantly bound in HA-KT mice compared with the control group (Fig. 3L).

HFD-fed Kcnq1ot1 truncated mice exhibit decreased pancreatic β-cell mass via increased C/EBPβ and Cdkn1c expression

The pathogenesis of T2DM is reported to be caused by both genetic and environmental factors [25]. If the KCNQ1 gene mutation is a genetic factor, then a typical environmental factor would be diet. Although we have previously shown that C/EBPβ accumulates in the islets of diabetic model mice [15], it was not clear whether C/EBPβ expression is altered in the islets of HFD-loaded mice without elevated blood glucose levels. In the present study, we confirmed the expression level by immunoblot and real-time PCR, and found that C/EBPβ expression was upregulated in the islets of HFD-fed mice at both the protein and mRNA levels (Fig. 4A and B). Kcnq1ot1 truncated TP mice showed a 40% decrease in pancreatic β-cell volume compared with WT mice fed a normal chow diet (NCD) (Fig. 4C) [7], while TP fed a HFD showed an enhanced rate of decrease in pancreatic β-cell volume, specifically, a 60% decrease (Fig. 4C). Because Cdkn1c is thought to be a causative molecule for the decrease in pancreatic β-cell volume, we confirmed the expression level of Cdkn1c in pancreatic islets. Cdkn1c expression was upregulated in the islets of TP fed a NCD compared with WT mice, but Cdkn1c expression was further increased in the islets of TP fed a HFD (Fig. 4D). Ki67 staining revealed that the proliferative potential of HFD-fed TP islets was markedly decreased compared with the control group. Ki67 staining revealed that the islet proliferative capacity of HFD-fed TP islets was markedly decreased compared with the control group (Fig. 4E). TUNEL staining also showed increased apoptosis in the islets of TP mice fed a HFD, suggesting that the amount of pancreatic β-cells was decreased by these mechanisms (Fig. 4F). Finally, we analyzed insulin secretion using isolated islets from HFD-loaded TP and WT mice. Static incubation of pancreatic islets isolated from TP mice revealed no difference in the degree of basal or stimulated insulin secretion compared with islets from WT mice (Fig. 4G).

Fig. 4.

Pancreatic β-cell mass in high-fat diet-fed truncated paternal (TP) mice was reduced by increased cyclin dependent kinase inhibitor 1C (Cdkn1c) expression via CCAAT/enhancer binding protein beta (C/EBPβ) expression. (A) Immunoblot analysis of C/EBPβ expression in pancreatic islets of high-fat diet (HFD)-fed wild-type (WT) mice. Representative (left) and quantitative (right) data are shown. (B) Relative expression of C/EBPβ mRNA in islets isolated from WT and TP with normal chow diet (NCD) or HFD. (C) Immunostaining of insulin (red) and glucagon (green) in pancreatic sections from WT and TP with NCD or HFD. Quantitative (left) and representative (right) data are shown (scale bars: 50 μm). (D) Relative expression of Cdkn1c mRNA in islets isolated from WT and TP with NCD or HFD. (E) Percentage of Ki67+ β-cells per islet in each 10-week-old mouse. (F) The percentage of apoptotic β-cells per islet in each mouse was determined using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. (G) Insulin secretion by pancreatic islets isolated from 10-week-old HFD-fed WT and TP mice and incubated for 30 minutes with 2.8 or 16.7 mM glucose. Data were normalized by islet insulin content. All quantitative data are mean±standard error of the mean for (A) 5, (B) 5, (C) 4–5, (D) 7, (E) 5, (F) 3–6, or (G) 6 mice in each group. NS, not significant. ^aP<0.05, ^bP<0.01.

Decreased pancreatic β-cell mass in HFD-loaded Kcnq1ot1 truncated mice is restored by Cdkn1c deletion

It is clear that when Cdkn1c expression is increased in pancreatic β-cells, apoptosis is induced in pancreatic β-cells, resulting in a decrease in pancreatic β-cell volume. However, the effect of decreased Cdkn1c expression in pancreatic β-cells is not well understood. To investigate whether the decreased Cdkn1c expression in pancreatic β-cells affects pancreatic β-cell volume and glucose metabolism, we generated and analyzed pancreatic β-cell–specific Cdkn1c KO mice. Cdkn1c is an imprinted gene and is expressed only from maternal alleles after birth [26]. Therefore, we classified pancreatic β-cell–specific Cdkn1c KO mice into maternal Cdkn1c heterozygous KO mice (mKO), in which only the maternal allele lacks Cdkn1c, and paternal Cdkn1c heterozygous KO mice (pKO), in which only the paternal allele lacks Cdkn1c. Ins-Cre mice were used as controls for the analysis. Blood glucose and body weight were measured when the mice were fed a NCD, and no changes were observed among the three groups at the age of 8 weeks (Fig. 5A and B). In addition, glucose levels and body weights were also measured in each group fed a HFD, and no significant differences were found among the three groups (Fig. 5C and D). Serum insulin levels in each group were also compared when the mice were fed a NCD and a HFD, but again no differences were found under either condition (Fig. 5E). To confirm the expression level of Cdkn1c in islets, Western blotting was performed using isolated islets from each mouse, and the results showed that Cdkn1c expression was decreased in mKO (Fig. 5F). Next, the amount of pancreatic β-cells in each group was measured when the mice were fed a NCD, and no significant differences were found among the groups (Fig. 5G). Furthermore, pancreatic β-cell volume was also measured in the three groups fed a HFD, and again no significant differences were found (Fig. 5H). These results may be due to the fact that other cell cycle inhibitors compensate for Cdkn1c deficiency. To confirm that the decrease in pancreatic β-cell volume in TP was due to increased Cdkn1c expression, we subsequently generated TP with Cdkn1c heterozygous KO mice (CKO), in which Cdkn1c in the maternal allele of TP mice was deleted. CKO with reduced Cdkn1c expression in the pancreatic β-cells of TP showed recovery of pancreatic β-cell volume both at the birth stage or with a HFD (Fig. 6A and B). Finally, we confirmed proliferation and apoptosis in the pancreatic β-cells of these mice. Ki67, which was decreased in TP pancreatic β-cells, was restored to the same level as WT in CKO, while TUNEL-positive pancreatic β-cells, which were increased in TP, were predominantly decreased in CKO (Fig. 6C and D).

Fig. 5.

Analysis of metabolic data and pancreatic β-cell mass in pancreatic β-cell–specific cyclin dependent kinase inhibitor 1C (Cdkn1c) knockout mice. (A) Blood glucose levels in pancreatic β-cells of paternal Cdkn1c heterozygous knockout (pKO) mice, maternal Cdkn1c heterozygous knockout (mKO) mice in pancreatic β-cells, and Ins-Cre mice fed a normal chow diet (NCD) at the age of 8 weeks. (B) Body weight for each mouse fed NCD at the age of 8 weeks. (C) Blood glucose levels for each mouse fed a high-fat diet (HFD) at the age of 10 weeks. (D) Body weight for each mouse fed a HFD at the age of 8 weeks. (E) Serum insulin concentrations in mice fed a NCD at the age of 8 weeks (left) or HFD at the age of 10 weeks (right). (F) Immunoblot analysis of CDKN1C expression in islets isolated from each mouse. (G, H) Immunostaining of insulin (red) and glucagon (green) in pancreatic sections from each mouse fed a NCD (G) or HFD (H). Quantitative (left) and representative (right) data are shown (scale bars: 50 μm). All quantitative data are mean±standard error of the mean for (A) 10, (B) 13–14, (C) 8, (D) 12, (E) 6–7, (G) 5, or (H) 6–9 mice in each group. NS, not significant.

Fig. 6.

Effect of reduced expression of cyclin dependent kinase inhibitor 1C (Cdkn1c) in pancreatic β-cells of KCNQ1 opposite strand/antisense transcript 1 (Kcnq1ot1) truncated mice on pancreatic β-cell mass. (A, B) Immunostaining of insulin (red) and glucagon (green) in pancreatic sections from wild-type (WT), truncated paternal (TP), and TP with Cdkn1c heterozygous knockout mice (CKO) at the birth stage (A) and with a high-fat diet (HFD) (B). Quantitative (left) and representative (right) data are shown (scale bars: 50 μm). (C) Percentage of Ki67+ β-cells per islet in each HFD-fed mouse. (D) Percentage of apoptotic β-cells per islet in each HFD-fed mouse was determined using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. (E) Model of the mechanism of pancreatic β-cell mass reduction by the interaction of reduced Kcnq1ot1 expression and environmental factors. All quantitative data are mean±standard error of the mean for (A) 4–5, (B) 4–5, (C) 3–6, or (D) 5 mice in each group. ER, endoplasmic reticulum; GRP78, glucose-regulated protein 78. ^aP<0.05, ^bP<0.01.

DISCUSSION

The global population of patients with T2DM continues to increase, exerting negative impacts on the quality of life and life expectancy for each patient as well as creating economic problems for society due to rising healthcare costs [27,28]. The pathogenesis of many diseases is believed to be due to genetic factors, environmental factors, or both. In T2DM in particular, both of these factors have a significant influence on pathogenesis, and the ratio of their contribution varies from patient to patient. Therefore, this study was conducted to elucidate the mechanism by which these factors exert a synergistic effect instead of an additive one.

Previous GWASs have reported numerous susceptibility genes for T2DM [29,30]. Given that the pathogenesis of diabetes differs significantly between Asians and Caucasians, GWASs should be performed for each ethnic group. Many previous GWASs have mixed Caucasian and Asian samples, with Caucasian samples accounting for the dominant share. With this in mind, meta-analyses conducted according to ancestry group have revealed large differences in T2DM susceptibility genes among ethnic groups [31]. In recent years, some GWASs have been reported by ethnicity, and a GWAS on 400,000 East Asians was reported in 2020 [32]. The present study shows that certain signals can cause different pathologies in different tissues, reaffirming the importance of genetic factors in the pathogenesis of T2DM. In addition, recent reports have linked GWAS data to diabetes-induced cardiovascular disease and to cell-specific phenotypes, shedding light on the heterogeneity of the pathogenesis of T2DM [33]. Thus, the continued study of T2DM susceptibility genes is expected to lead to a better understanding of the pathophysiology of individual diabetes in the future.

KCNQ1 is a voltage-gated potassium channel that contributes to cellular repolarization [34]. In 2008, Yasuda et al. [1] reported that SNPs in the 15th intron of the KCNQ1 gene are associated with the development of T2DM in East Asians, including Japanese and Chinese. This result was subsequently replicated in a GWAS of Europeans [35], and many laboratories have also reported decreased insulin secretory capacity in groups with the corresponding risk allele [3,4]. We previously reported that KCNQ1 mutations in pancreatic β-cells upregulate expression of the cell cycle inhibitor CDKN1C via disruption of imprinting regulation [7]. Although it remains unclear whether there is a correlation between KCNQ1 SNP and KCNQ1OT1 or CDKN1C expression in human islets, it was previously reported that CDKN1C expression is enhanced in the risk allele of KCNQ1 SNP rs163184, using pancreatic beta cell lines in vitro [36]. In addition, although increased CDKN1C expression in pancreatic β-cells was found to decrease pancreatic β-cell volume, the decrease in pancreatic β-cell volume in Kcnq1ot1 truncation mice on a NCD was only about 30% and there was no significant difference in blood glucose levels under normal conditions [7]. If CDKN1C is a major contributor to the pathogenesis of diabetes, there may be additional mechanisms by which CDKN1C expression is increased. For example, we noted the presence of a CCAAT motif in the CDKN1C promoter.

The CCAAT motif is a binding motif of the C/EBP family of transcription factors, and C/EBPβ is reported to binds to the CCAAT motif in chondrocytes and induce differentiation via transcriptional activation of Cdkn1c [37]. It has been previously shown that C/EBPβ expression is upregulated in pancreatic β-cells under endoplasmic reticulum stress conditions [38]. Furthermore, we previously reported that accumulation of C/EBPβ in pancreatic β-cells inhibits adaptive unfolded protein response and induces apoptosis of pancreatic β-cells by downregulating glucose-regulated protein 78 (GRP78) expression [15]. In other words, C/EBPβ accumulation is thought to contribute to the decrease in pancreatic β-cell volume in diabetic pancreatic β-cells, and in fact, C/EBPβ expression is upregulated in pancreatic β-cells from T2DM patients [39]. Although it has been reported that C/EBPβ accumulates in pancreatic β-cells due to environmental factors, there have been no reports on the relationship between C/EBPβ and genetic factors of T2DM. We therefore hypothesized that C/EBPβ, which accumulates in pancreatic β-cells due to environmental factors such as a HFD and obesity, may affect CDKN1C expression via the KCNQ1 gene abnormality, which are genetic factors. Accordingly, we performed both in vitro and in vivo experiments to simultaneously induce decreased Kcnq1ot1 expression, which regulates imprinting, as well as C/EBPβ overexpression in the cells. In both experiments, the DNA demethylation state facilitated binding of C/EBPβ to the Cdkn1c promoter, indicating that it enhances Cdkn1c expression. We have previously reported that, in Kcnq1ot1-truncated mice, Cdkn1c expression is upregulated specifically in pancreatic β-cells in association with reduced Kcnq1ot1 expression [7]. Additionally, in the present study, we found that Cdkn1c expression was further upregulated in pancreatic β-cells of KT mice due to C/EBPβ overexpression. In the future, we would like to confirm this mechanism by conducting a recovery experiment on the phenotype by crossing pancreatic β-cell–specific Cdkn1c KO mice with KT mice.

Cdkn1c, which is also called p57, is a cell cycle inhibitor, and its increased expression is known to decrease pancreatic β-cell mass [8]. The results of the present may suggest that the risk of developing T2DM due to the presence of SNPs in the KCNQ1 gene may be synergistically increased by interaction with environmental factors (Fig. 6E).

Although diet and eating habits contribute substantially to environmental factors, there are not many reports on interactions with genetic factors. A GWAS of East Asians reported that polymorphisms in the aldehyde dehydrogenase 2 family member (ALDH2) gene, which encodes an enzyme related to alcohol metabolism, significantly increased the risk of developing T2DM only in men [32]. The ALDH2 gene polymorphism increases alcohol tolerance, and thus heavy alcohol use increases the risk of developing T2DM. This mechanism is thought to be due not only to increased caloric intake but also to reduced glucose clearance due to decreased insulin sensitivity in the liver caused by heavy alcohol intake [40]. In addition, it has been shown that DNA methylation in adipocytes is altered when diet-induced obesity such as that caused by a HFD is present [41]. This phenomenon has been observed in both humans and mice. Integrative analysis of DNA methylation with T2DM susceptibility genes identified by GWAS identified AKT serine/threonine kinase 2 (Akt2) and tumor necrosis factor alpha-induced protein 8-like 2 (Tnfaip8l2) as candidate molecules. These molecules are thought to contribute to the development of T2DM because they cause insulin resistance by suppressing its function in adipocytes. It is also well known that Tcf7l2 risk alleles increase the risk of developing T2DM, and associations with decreased insulin secretion and decreased incretin secretion have been reported as mechanisms [42,43]. A cohort study reported that the increased risk of developing diabetes due to Tcf7l2 was inversely associated with dietary fiber intake [44]. Solute carrier family 30 member 8 (SLC30A8) encodes a zinc transporter and was similarly identified as a susceptibility gene for T2DM by a GWAS [45], and carriers of the risk allele are reported to have elevated fasting blood glucose and decreased insulin secretion [46]. Mice lacking ZnT8, a protein encoded by Slc30a8, and overexpressing human islet amyloid polypeptide were fed a HFD and developed pancreatic β-cell failure [47]. The presence of mutations in T2DM susceptibility genes under metabolic stress is thought to dramatically increase the risk of developing diabetes.

SUPPLEMENTARY MATERIALS

Supplementary materials related to this article can be found online at https://doi.org/10.4093/dmj.2024.0790.

Supplementary Methods.dmj-2024-0790-Supplementary-Methods.pdf

Notes

CONFLICTS OF INTEREST

Yoshiaki Kido has been an International editorial board members of the Diabetes & Metabolism Journal since 2022. He was not in volved in the review process of this article. Otherwise, there was no conflict of interest.

AUTHOR CONTRIBUTIONS

Conception or design: S.A., H.I., M.M., H.I., K.I.N., W.O., M.K., Y.K.

Acquisition, analysis, or interpretation of data: all authors.

Drafting the work or revising: S.A., H.I., M.M., H.I., M.K., Y.K.

Final approval of the manuscript: S.A., M.K., Y.K.

FUNDING

This research was supported by Grants-in-Aid for Creative Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (17H01966 and 21K08579 to Yoshiaki Kido; 17K09882, 18KK0438, and 20K08860 to Shunichiro Asahara; 20K08906 and 23K07990 to Maki Kimura-Koyanagi; and 20K17535 and 22K08653 to Ayumi Kanno). The funders had no role in study design, data collections and analysis, decision to publish, or preparation of the manuscript.

ACKNOWLEDGMENTS

We thank H. Etoh, A. Tanizawa, and K. Miyazaki for technical assistance.

References

1. Yasuda K, Miyake K, Horikawa Y, Hara K, Osawa H, Furuta H, et al. Variants in KCNQ1 are associated with susceptibility to type 2 diabetes mellitus. Nat Genet 2008;40:1092–7.

2. Unoki H, Takahashi A, Kawaguchi T, Hara K, Horikoshi M, Andersen G, et al. SNPs in KCNQ1 are associated with susceptibility to type 2 diabetes in East Asian and European populations. Nat Genet 2008;40:1098–102.

3. Tan JT, Nurbaya S, Gardner D, Ye S, Tai ES, Ng DP. Genetic variation in KCNQ1 associates with fasting glucose and beta-cell function: a study of 3,734 subjects comprising three ethnicities living in Singapore. Diabetes 2009;58:1445–9.

4. Hu C, Wang C, Zhang R, Ma X, Wang J, Lu J, et al. Variations in KCNQ1 are associated with type 2 diabetes and beta cell function in a Chinese population. Diabetologia 2009;52:1322–5.

5. Wang Q, Curran ME, Splawski I, Burn TC, Millholland JM, VanRaay TJ, et al. Positional cloning of a novel potassium channel gene: KVLQT1 mutations cause cardiac arrhythmias. Nat Genet 1996;12:17–23.

6. Smilinich NJ, Day CD, Fitzpatrick GV, Caldwell GM, Lossie AC, Cooper PR, et al. A maternally methylated CpG island in KvLQT1 is associated with an antisense paternal transcript and loss of imprinting in Beckwith-Wiedemann syndrome. Proc Natl Acad Sci U S A 1999;96:8064–9.

7. Asahara S, Etoh H, Inoue H, Teruyama K, Shibutani Y, Ihara Y, et al. Paternal allelic mutation at the Kcnq1 locus reduces pancreatic β-cell mass by epigenetic modification of Cdkn1c. Proc Natl Acad Sci U S A 2015;112:8332–7.

8. Avrahami D, Li C, Yu M, Jiao Y, Zhang J, Naji A, et al. Targeting the cell cycle inhibitor p57Kip2 promotes adult human β cell replication. J Clin Invest 2014;124:670–4.

9. Cavalli G, Heard E. Advances in epigenetics link genetics to the environment and disease. Nature 2019;571:489–99.

10. Asahara SI, Inoue H, Kido Y. Regulation of pancreatic β-cell mass by gene-environment interaction. Diabetes Metab J 2022;46:38–48.

11. Koza RA, Nikonova L, Hogan J, Rim JS, Mendoza T, Faulk C, et al. Changes in gene expression foreshadow diet-induced obesity in genetically identical mice. PLoS Genet 2006;2e81.

12. Kim S, Moon S, Popkin BM. The nutrition transition in South Korea. Am J Clin Nutr 2000;71:44–53.

13. Wang Q, Jensen HH, Johnson SR. China’s nutrient availability and sources, 1950-1991. Food Policy 1993;18:403–13.

14. Shin JY, Fitzpatrick GV, Higgins MJ. Two distinct mechanisms of silencing by the KvDMR1 imprinting control region. EMBO J 2008;27:168–78.

15. Matsuda T, Kido Y, Asahara S, Kaisho T, Tanaka T, Hashimoto N, et al. Ablation of C/EBPbeta alleviates ER stress and pancreatic beta cell failure through the GRP78 chaperone in mice. J Clin Invest 2010;120:115–26.

16. Matsumoto A, Takeishi S, Kanie T, Susaki E, Onoyama I, Tateishi Y, et al. p57 is required for quiescence and maintenance of adult hematopoietic stem cells. Cell Stem Cell 2011;9:262–71.

17. Kawada Y, Asahara SI, Sugiura Y, Sato A, Furubayashi A, Kawamura M, et al. Histone deacetylase regulates insulin signaling via two pathways in pancreatic β cells. PLoS One 2017;12e0184435.

18. Bartolome A, Kimura-Koyanagi M, Asahara S, Guillen C, Inoue H, Teruyama K, et al. Pancreatic β-cell failure mediated by mTORC1 hyperactivity and autophagic impairment. Diabetes 2014;63:2996–3008.

19. Kanno A, Asahara SI, Furubayashi A, Masuda K, Yoshitomi R, Suzuki E, et al. GCN2 regulates pancreatic β cell mass by sensing intracellular amino acid levels. JCI Insight 2020;5e128820.

20. Asahara S, Shibutani Y, Teruyama K, Inoue HY, Kawada Y, Etoh H, et al. Ras-related C3 botulinum toxin substrate 1 (RAC1) regulates glucose-stimulated insulin secretion via modulation of F-actin. Diabetologia 2013;56:1088–97.

21. Redrup L, Branco MR, Perdeaux ER, Krueger C, Lewis A, Santos F, et al. The long noncoding RNA Kcnq1ot1 organises a lineage-specific nuclear domain for epigenetic gene silencing. Development 2009;136:525–30.

22. Mohammad F, Mondal T, Guseva N, Pandey GK, Kanduri C. Kcnq1ot1 noncoding RNA mediates transcriptional gene silencing by interacting with Dnmt1. Development 2010;137:2493–9.

23. Cucciolla V, Borriello A, Criscuolo M, Sinisi AA, Bencivenga D, Tramontano A, et al. Histone deacetylase inhibitors upregulate p57Kip2 level by enhancing its expression through Sp1 transcription factor. Carcinogenesis 2008;29:560–7.

24. Henriquez B, Hepp M, Merino P, Sepulveda H, van Wijnen AJ, Lian JB, et al. C/EBPβ binds the P1 promoter of the Runx2 gene and up-regulates Runx2 transcription in osteoblastic cells. J Cell Physiol 2011;226:3043–52.

25. Geng T, Huang T. Gene-environment interactions and type 2 diabetes. Asia Pac J Clin Nutr 2020;29:220–6.

26. Hatada I, Mukai T. Genomic imprinting of p57KIP2, a cyclindependent kinase inhibitor, in mouse. Nat Genet 1995;11:204–6.

27. Chew NW, Ng CH, Tan DJ, Kong G, Lin C, Chin YH, et al. The global burden of metabolic disease: data from 2000 to 2019. Cell Metab 2023;35:414–28.

28. Afroz A, Alramadan MJ, Hossain MN, Romero L, Alam K, Magliano DJ, et al. Cost-of-illness of type 2 diabetes mellitus in low and lower-middle income countries: a systematic review. BMC Health Serv Res 2018;18:972.

29. Walker JT, Saunders DC, Rai V, Chen HH, Orchard P, Dai C, et al. Genetic risk converges on regulatory networks mediating early type 2 diabetes. Nature 2023;624:621–9.

30. Halama A, Zaghlool S, Thareja G, Kader S, Al Muftah W, Mook-Kanamori M, et al. A roadmap to the molecular human linking multiomics with population traits and diabetes subtypes. Nat Commun 2024;15:7111.

31. Mahajan A, Spracklen CN, Zhang W, Ng MC, Petty LE, Kitajima H, et al. Multi-ancestry genetic study of type 2 diabetes highlights the power of diverse populations for discovery and translation. Nat Genet 2022;54:560–72.

32. Spracklen CN, Horikoshi M, Kim YJ, Lin K, Bragg F, Moon S, et al. Identification of type 2 diabetes loci in 433,540 East Asian individuals. Nature 2020;582:240–5.

33. Suzuki K, Hatzikotoulas K, Southam L, Taylor HJ, Yin X, Lorenz KM, et al. Genetic drivers of heterogeneity in type 2 diabetes pathophysiology. Nature 2024;627:347–57.

34. Barhanin J, Lesage F, Guillemare E, Fink M, Lazdunski M, Romey G. K(V)LQT1 and lsK (minK) proteins associate to form the I(Ks) cardiac potassium current. Nature 1996;384:78–80.

35. Voight BF, Scott LJ, Steinthorsdottir V, Morris AP, Dina C, Welch RP, et al. Twelve type 2 diabetes susceptibility loci identified through large-scale association analysis. Nat Genet 2010;42:579–89.

36. Hiramoto M, Udagawa H, Ishibashi N, Takahashi E, Kaburagi Y, Miyazawa K, et al. A type 2 diabetes-associated SNP in KCNQ1 (rs163184) modulates the binding activity of the locus for Sp3 and Lsd1/Kdm1a, potentially affecting CDKN1C expression. Int J Mol Med 2018;41:717–28.

37. Hirata M, Kugimiya F, Fukai A, Ohba S, Kawamura N, Ogasawara T, et al. C/EBPbeta promotes transition from proliferation to hypertrophic differentiation of chondrocytes through transactivation of p57. PLoS One 2009;4e4543.

38. Oh YS, Lee YJ, Kang Y, Han J, Lim OK, Jun HS. Exendin-4 inhibits glucolipotoxic ER stress in pancreatic β cells via regulation of SREBP1c and C/EBPβ transcription factors. J Endocrinol 2013;216:343–52.

39. Mizukami H, Takahashi K, Inaba W, Tsuboi K, Osonoi S, Yoshida T, et al. Involvement of oxidative stress-induced DNA damage, endoplasmic reticulum stress, and autophagy deficits in the decline of β-cell mass in Japanese type 2 diabetic patients. Diabetes Care 2014;37:1966–74.

40. Takeno K, Tamura Y, Kakehi S, Kaga H, Kawamori R, Watada H. ALDH2 rs671 is associated with elevated FPG, reduced glucose clearance and hepatic insulin resistance in Japanese men. J Clin Endocrinol Metab 2021;106:e3573–81.

41. Multhaup ML, Seldin MM, Jaffe AE, Lei X, Kirchner H, Mondal P, et al. Mouse-human experimental epigenetic analysis unmasks dietary targets and genetic liability for diabetic phenotypes. Cell Metab 2015;21:138–49.

42. Schafer SA, Tschritter O, Machicao F, Thamer C, Stefan N, Gallwitz B, et al. Impaired glucagon-like peptide-1-induced insulin secretion in carriers of transcription factor 7-like 2 (TCF7L2) gene polymorphisms. Diabetologia 2007;50:2443–50.

43. Færch K, Pilgaard K, Knop FK, Hansen T, Pedersen O, Jorgensen T, et al. Incretin and pancreatic hormone secretion in Caucasian non-diabetic carriers of the TCF7L2 rs7903146 risk T allele. Diabetes Obes Metab 2013;15:91–5.

44. Hindy G, Sonestedt E, Ericson U, Jing XJ, Zhou Y, Hansson O, et al. Role of TCF7L2 risk variant and dietary fibre intake on incident type 2 diabetes. Diabetologia 2012;55:2646–54.

45. Sladek R, Rocheleau G, Rung J, Dina C, Shen L, Serre D, et al. A genome-wide association study identifies novel risk loci for type 2 diabetes. Nature 2007;445:881–5.

46. Haupt A, Staiger H, Schafer SA, Kirchhoff K, Guthoff M, Machicao F, et al. The risk allele load accelerates the age-dependent decline in beta cell function. Diabetologia 2009;52:457–62.

47. Xu J, Wijesekara N, Regeenes R, Rijjal DA, Piro AL, Song Y, et al. Pancreatic β cell-selective zinc transporter 8 insufficiency accelerates diabetes associated with islet amyloidosis. JCI Insight 2021;6e143037.

Article information Continued

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.