Identification of a Novel β-Cell Glucokinase (GCK) Promoter Mutation (−71G>C) That Modulates GCK Gene Expression Through Loss of Allele-Specific Sp1 Binding Causing Mild Fasting Hyperglycemia in Humans

OBJECTIVE: Inactivating mutations in glucokinase (GCK) cause mild fasting hyperglycemia. Identification of a GCK mutation has implications for treatment and prognosis; therefore, it is important to identify these individuals. A significant number of patients have a phenotype suggesting a defect in glucokinase but no abnormality of GCK. We hypothesized that the GCK beta-cell promoter region, which currently is not routinely screened, could contain pathogenic mutations; therefore, we sequenced this region in 60 such probands. RESEARCH DESIGN AND METHODS: The beta-cell GCK promoter was sequenced in patient DNA. The effect of the identified novel mutation on GCK promoter activity was assessed using a luciferase reporter gene expression system. Electrophoretic mobility shift assays (EMSAs) were used to determine the impact of the mutation on Sp1 binding. RESULTS: A novel -71G>C mutation was identified in a nonconserved region of the human promoter sequence in six apparently unrelated probands. Family testing established cosegregation with fasting hyperglycemia (> or = 5.5 mmol/l) in 39 affected individuals. Haplotype analysis in the U.K. family and four of the Slovakian families demonstrated that the mutation had arisen independently. The mutation maps to a potential transcriptional activator binding site for Sp1. Reporter assays demonstrated that the mutation reduces promoter activity by up to fourfold. EMSAs demonstrated a dramatic reduction in Sp1 binding to the promoter sequence corresponding to the mutant allele. CONCLUSIONS: A novel beta-cell GCK promoter mutation was identified that significantly reduces gene expression in vitro through loss of regulation by Sp1. To ensure correct diagnosis of potential GCK-MODY (maturity-onset diabetes of the young) cases, analysis of the beta-cell GCK promoter should be included

Type 2 Diabetes Susceptibility Gene TCF7L2 and Its Role in β-Cell Function

Type 2 diabetes is associated with impaired insu-lin secretion. Both 1st- and 2nd-phase insulinsecretion are reduced, but the effect is particu-larly pronounced for the 1st phase. The pro-cesses culminating in impaired insulin secretion are not fully understood, but both genetic and environmental factors are thought to play a role. Over the past 2 years, genome-wide association scans have transformed the ge-netic landscape of type 2 diabetes susceptibility, with the current gene count close to 20 (1). A couple of common themes have emerged from these studies. First, the major-ity of the genes identified thus far seem to affect diabetes susceptibility through -cell dysfunction (2). Second, the risk alleles tend to be common in the population, but their effect on diabetes risk is relatively small (3,4). TCF7L2, the susceptibility gene with the largest effect on disease susceptibility discovered to date, was iden-tified pre–genome-wide association by Grant et al. i

Loss of AMP-activated protein kinase alpha 2 subunit in mouse beta-cells impairs glucose-stimulated insulin secretion and inhibits their sensitivity to hypoglycaemia

AMPK (AMP-activated protein kinase) signalling plays a key role in whole-body energy homoeostasis, although its precise role in pancreatic β-cell function remains unclear. In the present stusy, we therefore investigated whether AMPK plays a critical function in β-cell glucose sensing and is required for the maintenance of normal glucose homoeostasis. Mice lacking AMPKα2 in β-cells and a population of hypothalamic neurons (RIPCreα2KO mice) and RIPCreα2KO mice lacking AMPKα1 (α1KORIPCreα2KO) globally were assessed for whole-body glucose homoeostasis and insulin secretion. Isolated pancreatic islets from these mice were assessed for glucose-stimulated insulin secretion and gene expression changes. Cultured β-cells were examined electrophysiologically for their electrical responsiveness to hypoglycaemia. RIPCreα2KO mice exhibited glucose intolerance and impaired GSIS (glucose-stimulated insulin secretion) and this was exacerbated in α1KORIPCreα2KO mice. Reduced glucose concentrations failed to completely suppress insulin secretion in islets from RIPCreα2KO and α1KORIPCreα2KO mice, and conversely GSIS was impaired. β-Cells lacking AMPKα2 or expressing a kinase-dead AMPKα2 failed to hyperpolarize in response to low glucose, although KATP (ATP-sensitive potassium) channel function was intact. We could detect no alteration of GLUT2 (glucose transporter 2), glucose uptake or glucokinase that could explain this glucose insensitivity. UCP2 (uncoupling protein 2) expression was reduced in RIPCreα2KO islets and the UCP2 inhibitor genipin suppressed low-glucose-mediated wild-type mouse β-cell hyperpolarization, mimicking the effect of AMPKα2 loss. These results show that AMPKα2 activity is necessary to maintain normal pancreatic β-cell glucose sensing, possibly by maintaining high β-cell levels of UCP2

Tooth Discoloration in Patients With Neonatal Diabetes After Transfer Onto Glibenclamide: A previously unreported side effect

PublishedJournal ArticleMulticenter StudyResearch Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov'tOBJECTIVE To assess if tooth discoloration is a novel side effect of sulfonylurea therapy in patients with permanent neonatal diabetes due to mutations in KCNJ11. RESEARCH DESIGN AND METHODS A total of 67 patients with a known KCNJ11 mutation who had been successfully transferred from insulin injections onto oral sulfonylureas were contacted and asked about the development of tooth discoloration after transfer. RESULTS Altered tooth appearance was identified in 5 of the 67 patients. This was variable in severity, ranging from mild discoloration/staining (n = 4) to loss of enamel (n = 1) and was only seen in patients taking glibenclamide (glyburide). CONCLUSIONS These previously unreported side effects may relate to the developing tooth and/or to the high local concentrations in the children who frequently chewed glibenclamide tablets or took it as a concentrated solution. Given the multiple benefits of sulfonylurea treatment for patients with activating KCNJ11 mutations, this association warrants further investigation but should not preclude such treatment.This work was funded by the Welcome Trust (grant 067463/Z/2/Z), National Institutes of Health Grants DK-44752 and DK-20595, and a gift from the Kovler Family Foundation. S.E.F. is the Sir Graham Wilkins, Peninsula Medical School Research Fellow. A.T.H. is a Welcome Trust Research Leave Fellow. O.R.-C. was supported by an “Ayuda para contratos post-Formacio´n Sanitaria Especializada” from the “Instituto de Salud Carlos III” (FIS CM06/00013

TIGER : The gene expression regulatory variation landscape of human pancreatic islets

Genome-wide association studies (GWASs) identified hundreds of signals associated with type 2 diabetes (T2D). To gain insight into their underlying molecular mechanisms, we have created the translational human pancreatic islet genotype tissue-expression resource (TIGER), aggregating >500 human islet genomic datasets from five cohorts in the Horizon 2020 consortium T2DSystems. We impute genotypes using four reference panels and meta-analyze cohorts to improve the coverage of expression quantitative trait loci (eQTL) and develop a method to combine allele-specific expression across samples (cASE). We identify >1 million islet eQTLs, 53 of which colocalize with T2D signals. Among them, a low-frequency allele that reduces T2D risk by half increases CCND2 expression. We identify eight cASE colocalizations, among which we found a T2D-associated SLC30A8 variant. We make all data available through the TIGER portal (http://tiger.bsc.es), which represents a comprehensive human islet genomic data resource to elucidate how genetic variation affects islet function and translates into therapeutic insight and precision medicine for T2D.Peer reviewe

Effects of body weight and alcohol consumption on insulin sensitivity

<p>Abstract</p> <p>Background</p> <p>Obesity is a risk factor for the development of insulin resistance, which can eventually lead to type-2 diabetes. Alcohol consumption is a protective factor against insulin resistance, and thus protects against the development of type-2 diabetes. The mechanism by which alcohol protects against the development of type-2 diabetes is not well known. To determine the mechanism by which alcohol improves insulin sensitivity, we fed water or alcohol to lean, control, and obese mice. The aim of this study was to determine whether alcohol consumption and body weights affect overlapping metabolic pathways and to identify specific target genes that are regulated in these pathways.</p> <p>Method</p> <p>Adipose tissue dysfunction has been associated with the development of type-2 diabetes. We assessed possible gene expression alterations in epididymal white adipose tissue (WAT). We obtained WAT from mice fed a calorie restricted (CR), low fat (LF Control) or high fat (HF) diets and either water or 20% ethanol in the drinking water. We screened the expression of genes related to the regulation of energy homeostasis and insulin regulation using a gene array composed of 384 genes.</p> <p>Results</p> <p>Obesity induced insulin resistance and calorie restriction and alcohol improved insulin sensitivity. The insulin resistance in obese mice was associated with the increased expression of inflammatory markers Cd68, Il-6 and Il-1α; in contrast, most of these genes were down-regulated in CR mice. Anti-inflammatory factors such as Il-10 and adrenergic beta receptor kinase 1 (Adrbk1) were decreased in obese mice and increased by CR and alcohol. Also, we report a direct correlation between body weight and the expression of the following genes: Kcnj11 (potassium inwardly-rectifying channel, subfamily J, member 11), Lpin2 (lipin2), and Dusp9 (dual-specificity MAP kinase phosphatase 9).</p> <p>Conclusion</p> <p>We show that alcohol consumption increased insulin sensitivity. Additionally, alterations in insulin sensitivity related with obesity were coupled with alterations in inflammatory genes. We provide evidence that alcohol may improve insulin sensitivity by up-regulating anti-inflammatory genes. Moreover, we have indentified potential gene targets in energy metabolic pathways and signal transducers that may contribute to obesity-related insulin resistance as well as calorie restriction and alcohol-induced insulin sensitivity.</p

Disease risk prediction with rare and common variants

A number of studies have been conducted to investigate the predictive value of common genetic variants for complex diseases. To date, these studies have generally shown that common variants have no appreciable added predictive value over classical risk factors. New sequencing technology has enhanced the ability to identify rare variants that may have larger functional effects than common variants. One would expect rare variants to improve the discrimination power for disease risk by permitting more detailed quantification of genetic risk. Using the Genetic Analysis Workshop 17 simulated data sets for unrelated individuals, we evaluate the predictive value of rare variants by comparing prediction models built using the support vector machine algorithm with or without rare variants. Empirical results suggest that rare variants have appreciable effects on disease risk prediction

Type 2 Diabetes Susceptibility Gene Expression in Normal or Diabetic Sorted Human Alpha and Beta Cells: Correlations with Age or BMI of Islet Donors

BACKGROUND: Genome-wide association studies have identified susceptibility genes for development of type 2 diabetes. We aimed to examine whether a subset of these (comprising FTO, IDE, KCNJ11, PPARG and TCF7L2) were transcriptionally restricted to or enriched in human beta cells by sorting islet cells into alpha and beta - specific fractions. We also aimed to correlate expression of these transcripts in both alpha and beta cell types with phenotypic traits of the islet donors and to compare diabetic and non-diabetic cells. METHODOLOGY/PRINCIPAL FINDINGS: Islet cells were sorted using a previously published method and RNA was extracted, reverse transcribed and used as the template for quantitative PCR. Sorted cells were also analysed for insulin and glucagon immunostaining and insulin secretion from the beta cells as well as insulin, glucagon and GLP-1 content. All five genes were expressed in both alpha and beta cells, with significant enrichment of KCNJ11 in the beta cells and of TCF7L2 in the alpha cells. The ratio of KCNJ11 in beta to alpha cells was negatively correlated with BMI, while KCNJ11 expression in alpha cells was negatively correlated with age but not associated with BMI. Beta cell expression of glucagon, TCF7L2 and IDE was increased in cells from islets that had spent more time in culture prior to cell sorting. In beta cells, KCNJ11, FTO and insulin were positively correlated with each other. Diabetic alpha and beta cells had decreased expression of insulin, glucagon and FTO. CONCLUSIONS/SIGNIFICANCE: This study has identified novel patterns of expression of type 2 diabetes susceptibility genes within sorted islet cells and suggested interactions of gene expression with age or BMI of the islet donors. However, expression of these genes in islets is less associated with BMI than has been found for other tissues