A gene-driven approach to the identification of ENU mutants in the mouse.

The construction of parallel archives of DNA and sperm from mice mutagenized with ethylnitrosurea (ENU) represents a potentially powerful and rapid approach for identifying point mutations in any gene in the mouse genome. We provide support for this approach and report the identification of mutations in the gene (Gjb2) encoding connexin 26, using archives established from the UK ENU mutagenesis program.

Expression of an activating mutation in the gene encoding the KATP channel subunit Kir6.2 in mouse pancreatic beta cells recapitulates neonatal diabetes.

Neonatal diabetes is a rare monogenic form of diabetes that usually presents within the first six months of life. It is commonly caused by gain-of-function mutations in the genes encoding the Kir6.2 and SUR1 subunits of the plasmalemmal ATP-sensitive K+ (KATP) channel. To better understand this disease, we generated a mouse expressing a Kir6.2 mutation (V59M) that causes neonatal diabetes in humans and we used Cre-lox technology to express the mutation specifically in pancreatic beta cells. These beta-V59M mice developed severe diabetes soon after birth, and by 5 weeks of age, blood glucose levels were markedly increased and insulin was undetectable. Islets isolated from beta-V59M mice secreted substantially less insulin and showed a smaller increase in intracellular calcium in response to glucose. This was due to a reduced sensitivity of KATP channels in pancreatic beta cells to inhibition by ATP or glucose. In contrast, the sulfonylurea tolbutamide, a specific blocker of KATP channels, closed KATP channels, elevated intracellular calcium levels, and stimulated insulin release in beta-V59M beta cells, indicating that events downstream of KATP channel closure remained intact. Expression of the V59M Kir6.2 mutation in pancreatic beta cells alone is thus sufficient to recapitulate the neonatal diabetes observed in humans. beta-V59M islets also displayed a reduced percentage of beta cells, abnormal morphology, lower insulin content, and decreased expression of Kir6.2, SUR1, and insulin mRNA. All these changes are expected to contribute to the diabetes of beta-V59M mice. Their cause requires further investigation.

Author Correction: Regulatory variants at KLF14 influence type 2 diabetes risk via a female-specific effect on adipocyte size and body composition.

In the version of this article originally published, minus signs were missing from the three ß-values for BMI given in Table 1. The errors have been corrected in the HTML and PDF versions of the article.

Regulatory variants at KLF14 influence type 2 diabetes risk via a female-specific effect on adipocyte size and body composition.

Individual risk of type 2 diabetes (T2D) is modified by perturbations to the mass, distribution and function of adipose tissue. To investigate the mechanisms underlying these associations, we explored the molecular, cellular and whole-body effects of T2D-associated alleles near KLF14. We show that KLF14 diabetes-risk alleles act in adipose tissue to reduce KLF14 expression and modulate, in trans, the expression of 385 genes. We demonstrate, in human cellular studies, that reduced KLF14 expression increases pre-adipocyte proliferation but disrupts lipogenesis, and in mice, that adipose tissue-specific deletion of Klf14 partially recapitulates the human phenotype of insulin resistance, dyslipidemia and T2D. We show that carriers of the KLF14 T2D risk allele shift body fat from gynoid stores to abdominal stores and display a marked increase in adipocyte cell size, and that these effects on fat distribution, and the T2D association, are female specific. The metabolic risk associated with variation at this imprinted locus depends on the sex both of the subject and of the parent from whom the risk allele derives.

Deletion of nicotinamide nucleotide transhydrogenase: a new quantitive trait locus accounting for glucose intolerance in C57BL/6J mice.

The C57BL/6J mouse displays glucose intolerance and reduced insulin secretion. The genetic locus underlying this phenotype was mapped to nicotinamide nucleotide transhydrogenase (Nnt) on mouse chromosome 13, a nuclear-encoded mitochondrial protein involved in beta-cell mitochondrial metabolism. C57BL/6J mice have a naturally occurring in-frame five-exon deletion in Nnt that removes exons 7-11. This results in a complete absence of Nnt protein in these mice. We show that transgenic expression of the entire Nnt gene in C57BL/6J mice rescues their impaired insulin secretion and glucose-intolerant phenotype. This study provides direct evidence that Nnt deficiency results in defective insulin secretion and inappropriate glucose homeostasis in male C57BL/6J mice.

Mutant Mice With Calcium-Sensing Receptor Activation Have Hyperglycemia That Is Rectified by Calcilytic Therapy.

The calcium-sensing receptor (CaSR) is a family C G-protein-coupled receptor that plays a pivotal role in extracellular calcium homeostasis. The CaSR is also highly expressed in pancreatic islet α- and ß-cells that secrete glucagon and insulin, respectively. To determine whether the CaSR may influence systemic glucose homeostasis, we characterized a mouse model with a germline gain-of-function CaSR mutation, Leu723Gln, referred to as Nuclear flecks (Nuf). Heterozygous- (CasrNuf/+) and homozygous-affected (CasrNuf/Nuf) mice were shown to have hypocalcemia in association with impaired glucose tolerance and insulin secretion. Oral administration of a CaSR antagonist compound, known as a calcilytic, rectified the glucose intolerance and hypoinsulinemia of CasrNuf/+ mice and ameliorated glucose intolerance in CasrNuf/Nuf mice. Ex vivo studies showed CasrNuf/+ and CasrNuf/Nuf mice to have reduced pancreatic islet mass and ß-cell proliferation. Electrophysiological analysis of isolated CasrNuf/Nuf islets showed CaSR activation to increase the basal electrical activity of ß-cells independently of effects on the activity of the adenosine triphosphate (ATP)-sensitive K+ (KATP) channel. CasrNuf/Nuf mice also had impaired glucose-mediated suppression of glucagon secretion, which was associated with increased numbers of α-cells and a higher α-cell proliferation rate. Moreover, CasrNuf/Nuf islet electrophysiology demonstrated an impairment of α-cell membrane depolarization in association with attenuated α-cell basal KATP channel activity. These studies indicate that the CaSR activation impairs glucose tolerance by a combination of α- and ß-cell defects and also influences pancreatic islet mass. Moreover, our findings highlight a potential application of targeted CaSR compounds for modulating glucose metabolism.

RNA-sequencing of WFS1-deficient pancreatic islets.

Wolfram syndrome, an autosomal recessive disorder characterized by juvenile-onset diabetes mellitus and optic atrophy, is caused by mutations in theWFS1gene.WFS1encodes an endoplasmic reticulum resident transmembrane protein. TheWfs1-null mice exhibit progressive insulin deficiency and diabetes. The aim of this study was to describe the insulin secretion and transcriptome of pancreatic islets inWFS1-deficient mice.WFS1-deficient (Wfs1KO) mice had considerably less pancreatic islets than heterozygous (Wfs1HZ) or wild-type (WT) mice. Wfs1KOpancreatic islets secreted less insulin after incubation in 2 and 10 mmol/L glucose and with tolbutamide solution compared toWTand Wfs1HZislets, but not after stimulation with 20 mmol/L glucose. Differences in proinsulin amount were not statistically significant although there was a trend that Wfs1KOhad an increased level of proinsulin. After incubation in 2 mmol/L glucose solution the proinsulin/insulin ratio in Wfs1KOwas significantly higher than that ofWTand Wfs1HZRNA-seq from pancreatic islets found melastatin-related transient receptor potential subfamily member 5 protein gene (Trpm5) to be downregulated inWFS1-deficient mice. Functional annotation ofRNAsequencing results showed thatWFS1 deficiency influenced significantly the pathways related to tissue morphology, endocrine system development and function, molecular transport network.

Islet Insulin Secretion Measurements in the Mouse.

This article describes detailed protocols for in vitro measurements of insulin function and secretion in isolated mouse islets for the analysis of glucose homeostasis. We specify a method of enzyme digestion and hand picking to isolate and release the greatest number of high quality islets from the pancreas of the mouse. We describe an effective method for generating dynamic measurements of insulin secretion using a perifusion assay including a detailed protocol for constructing a peristaltic pump and tubing assembly. In addition we describe an alternative and simple technique for measuring insulin secretion using static incubation of isolated islets. © 2016 by John Wiley & Sons, Inc.

Increased Expression of the Diabetes Gene SOX4 Reduces Insulin Secretion by Impaired Fusion Pore Expansion.

The transcription factor Sox4 has been proposed to underlie the increased type 2 diabetes risk linked to an intronic single nucleotide polymorphism in CDKAL1 In a mouse model expressing a mutant form of Sox4, glucose-induced insulin secretion is reduced by 40% despite normal intracellular Ca(2+) signaling and depolarization-evoked exocytosis. This paradox is explained by a fourfold increase in kiss-and-run exocytosis (as determined by single-granule exocytosis measurements) in which the fusion pore connecting the granule lumen to the exterior expands to a diameter of only 2 nm, which does not allow the exit of insulin. Microarray analysis indicated that this correlated with an increased expression of the exocytosis-regulating protein Stxbp6. In a large collection of human islet preparations (n = 63), STXBP6 expression and glucose-induced insulin secretion correlated positively and negatively with SOX4 expression, respectively. Overexpression of SOX4 in the human insulin-secreting cell EndoC-ßH2 interfered with granule emptying and inhibited hormone release, the latter effect reversed by silencing STXBP6 These data suggest that increased SOX4 expression inhibits insulin secretion and increased diabetes risk by the upregulation of STXBP6 and an increase in kiss-and-run exocytosis at the expense of full fusion. We propose that pharmacological interventions promoting fusion pore expansion may be effective in diabetes therapy.

A new mouse model of type 2 diabetes, produced by N-ethyl-nitrosourea mutagenesis, is the result of a missense mutation in the glucokinase gene.

Here we report the first cloned N-ethyl-nitrosourea (ENU)-derived mouse model of diabetes. GENA348 was identified through free-fed plasma glucose measurement, being more than 2 SDs above the population mean of a cohort of >1,201 male ENU mutant mice. The underlying gene was mapped to the maturity-onset diabetes of the young (MODY2) homology region of mouse chromosome 11 (logarithm of odds 6.0). Positional candidate gene analyses revealed an A to T transversion mutation in exon 9 of the glucokinase gene, resulting in an isoleucine to phenylalanine change at amino acid 366 (I366F). Heterozygous mutants have 67% of the enzyme activity of wild-type littermates (P < 0.0012). Homozygous mutants have less enzyme activity (14% of wild-type activity) and are even less glucose tolerant. The GENA348 allele is novel because no mouse or human diabetes studies have described a mutation in the corresponding amino acid position. It is also the first glucokinase missense mutation reported in mice and is homozygous viable, unlike the global knockout mutations. This work demonstrates that ENU mutagenesis screens can be used to generate models of complex phenotypes, such as type 2 diabetes, that are directly relevant to human disease.

Loss of arylformamidase with reduced thymidine kinase expression leads to impaired glucose tolerance.

Tryptophan metabolites have been linked in observational studies with type 2 diabetes, cognitive disorders, inflammation and immune system regulation. A rate-limiting enzyme in tryptophan conversion is arylformamidase (Afmid), and a double knockout of this gene and thymidine kinase (Tk) has been reported to cause renal failure and abnormal immune system regulation. In order to further investigate possible links between abnormal tryptophan catabolism and diabetes and to examine the effect of single Afmid knockout, we have carried out metabolic phenotyping of an exon 2 Afmid gene knockout. These mice exhibit impaired glucose tolerance, although their insulin sensitivity is unchanged in comparison to wild-type animals. This phenotype results from a defect in glucose stimulated insulin secretion and these mice show reduced islet mass with age. No evidence of a renal phenotype was found, suggesting that this published phenotype resulted from loss of Tk expression in the double knockout. However, despite specifically removing only exon 2 of Afmid in our experiments we also observed some reduction of Tk expression, possibly due to a regulatory element in this region. In summary, our findings support a link between abnormal tryptophan metabolism and diabetes and highlight beta cell function for further mechanistic analysis.

Mutations in Mll2, an H3K4 methyltransferase, result in insulin resistance and impaired glucose tolerance in mice.

We employed a random mutagenesis approach to identify novel monogenic determinants of type 2 diabetes. Here we show that haplo-insufficiency of the histone methyltransferase myeloid-lineage leukemia (Mll2/Wbp7) gene causes type 2 diabetes in the mouse. We have shown that mice heterozygous for two separate mutations in the SET domain of Mll2 or heterozygous Mll2 knockout mice were hyperglycaemic, hyperinsulinaemic and developed non-alcoholic fatty liver disease. Consistent with previous Mll2 knockout studies, mice homozygous for either ENU mutation (or compound heterozygotes) died during embryonic development at 9.5-14.5 days post coitum. Heterozygous deletion of Mll2 induced in the adult mouse results in a normal phenotype suggesting that changes in chromatin methylation during development result in the adult phenotype. Mll2 has been shown to regulate a small subset of genes, a number of which Neurod1, Enpp1, Slc27a2, and Plcxd1 are downregulated in adult mutant mice. Our results demonstrate that histone H3K4 methyltransferase Mll2 is a component of the genetic regulation necessary for glucose homeostasis, resulting in a specific disease pattern linking chromatin modification with causes and progression of type 2 diabetes, providing a basis for its further understanding at the molecular level.

Insulin secretion from beta-cells is affected by deletion of nicotinamide nucleotide transhydrogenase.

Nicotinamide nucleotide transhydrogenase (NNT) is an inner mitochondrial membrane transmembrane protein involved in regenerating NADPH, coupled with proton translocation across the inner membrane. We have shown that a defect in Nnt function in the mouse, and specifically within the beta-cell, leads to a reduction in insulin secretion. This chapter describes methods for examining Nnt function in the mouse. This includes generating in vivo models with point mutations and expression of Nnt by transgenesis, and making in vitro models, by silencing of gene expression. In addition, techniques are described to measure insulin secretion, calcium and hydrogen peroxide concentrations, membrane potential, and NNT activity. These approaches and techniques can also be applied to other genes of interest.

Role of the transcription factor sox4 in insulin secretion and impaired glucose tolerance.

OBJECTIVES: To identify, map, clone, and functionally validate a novel mouse model for impaired glucose tolerance and insulin secretion. RESEARCH DESIGN AND METHODS: Haploinsufficiency of the insulin receptor and associated mild insulin resistance has been used to sensitize an N-ethyl-N-nitrosourea (ENU) screen to identify novel mutations resulting in impaired glucose tolerance and diabetes. The new impaired glucose tolerance 4 (IGT4) model was selected using an intraperitoneal glucose tolerance test and inheritance of the phenotype confirmed by generation of backcross progeny. Segregation of the phenotype was correlated with genotype information to map the location of the gene and candidates sequenced for mutations. The function of the SRY-related high mobility group (HMG)-box 4 (Sox4) gene in insulin secretion was tested using another ENU allele and by small interfering RNA silencing in insulinoma cells. RESULTS: We describe two allelic autosomal dominant mutations in the highly conserved HMG box of the transcription factor Sox4. Previously associated with pancreas development, Sox4 mutations in the adult mouse result in an insulin secretory defect, which exhibits impaired glucose tolerance in association with insulin receptor(+/-)-induced insulin resistance. Elimination of the Sox4 transcript in INS1 and Min6 cells resulted in the abolition of glucose-stimulated insulin release similar to that observed for silencing of the key metabolic enzyme glucokinase. Intracellular calcium measurements in treated cells indicate that this defect lies downstream of the ATP-sensitive K(+) channel (K(ATP) channel) and calcium influx. CONCLUSIONS: IGT4 represents a novel digenic model of insulin resistance coupled with an insulin secretory defect. The Sox4 gene has a role in insulin secretion in the adult beta-cell downstream of the K(ATP) channel.

ENU mutagenesis in the mouse: application to human genetic disease.

Genetic approaches in model organisms provide a powerful means by which to examine the biological basis of human diseases as well as the physiological processes that are affected by them. Although not without its drawbacks, the mouse has become the mammalian species of choice in studying the molecular basis of disease. Targeted mutagenesis approaches in the mouse have led to dramatic increases in our understanding of human disease processes. As a complement to these gene-driven studies, three developments have led to the reassessment of a phenotype-driven approach in the mouse--the accumulation of information that has emerged from human and mouse genome sequencing projects, the use of high-efficiency point mutagens such as N-ethyl-N-nitrosourea (ENU) and the application of systematic hierarchical screening protocols for the mouse. In this paper, progress with existing phenotypic screening programmes is discussed and opportunities for the development of new mouse disease models are presented.

Angiotensin-1-converting enzyme (ACE) plasma concentration is influenced by multiple ACE-linked quantitative trait nucleotides.

Circulating angiotensin-1-converting enzyme (ACE) is a highly heritable trait, and a major component of the genetic variance maps to the region of the ACE gene. The strong effect of the locus, and the interest in ACE as a candidate gene for cardiovascular disorders, has led to extensive investigation of its relationship to the ACE phenotype, providing one of the most complete examples of quantitative trait locus (QTL) analysis in humans. Resequencing of ACE followed by haplotype analysis in families of British and French origin has shown that the genetic variants that are primarily associated with the ACE trait map to an 18 kb interval flanked by two intragenic, ancestral recombination breakpoints. This critical interval contains dozens of ACE-associated variants in Caucasians, but identification of which of these directly influence ACE concentration is ambiguous because of the almost complete linkage disequilibrium in European populations. In a complementary sequencing and genotyping study of individuals from West African families, we show that this population has much greater haplotype diversity across the gene. Through analysis of the contrasting relationships of the trait phenotype with haplotypes that carry different allelic combinations from those observed in Caucasians, we demonstrate that (at least) two major intragenic sites within the critical interval and (at least) one minor promoter site are associated with the ACE quantitative trait through additive effects. These results point to the importance of analysing diverse populations with different gene genealogies in gene-association studies.

A gene-driven ENU-based approach to generating an allelic series in any gene.

N-ethyl-N-nitrosourea (ENU) introduces mutations throughout the mouse genome at relatively high efficiency. Successful high-throughput phenotype screens have been reported and alternative screens using sequence-based approaches have been proposed. For the purpose of generating an allelic series in selected genes by a sequence-based approach, we have constructed an archive of over 4000 DNA samples from individual F1 ENU-mutagenized mice paralleled by frozen sperm samples. Together with our previously reported archive, the total size now exceeds 6000 individuals. A gene-based screen of 27.4 Mbp of DNA, carried out using denaturing high-performance liquid chromatography (DHPLC), found a mutation rate of 1 in 1.01 Mbp of which 1 in 1.82 Mbp were potentially functional. Screening of whole or selected regions of genes on subsets of the archive has allowed us to identify 15 new alleles from 9 genes out of 15 tested. This is a powerful adjunct to conventional mutagenesis strategies and has the advantage of generating a variety of alleles with potentially different phenotypic outcomes that facilitate the investigation of gene function. It is now available to academic collaborators as a community resource.