Roles of the type 2 diabetes (T2D)-associated gene C2cd4a in regulating glucose homeostasis in the mouse

Lead Research Organisation: Imperial College London
Department Name: Dept of Medicine


Type 2 diabetes (T2D) is a strongly hereditary disease and studies over the past decade have highlighted a large number of regions in the human genome which affect disease risk. Identifying the causal gene(s) in each region is now an important goal and may provide targets for new therapies that treat the underlying disease aetiology (i.e. causes and progress), rather than the symptoms alone.

"Genome-wide association studies", carried out since 2007 and involving comparisons of the genetic variation in thousands of non-diabetic versus T2D patients, have identified almost 500 coding genes in >100 genomic loci as contributors to type 2 diabetes heredity. Studies in man have also indicated that the vast majority of these variants impact the secretion of insulin from the pancreas rather than the action of the hormone on target tissues (liver, muscle, etc). Our own recent studies, involving post mortem pancreatic islet material from patients, suggest that changes in the expression of genes at a locus on chromosome 15, namely VPS13C, C2CD4A and C2CD4B, may contribute to altered disease risk.

Mice deleted selectively in the pancreatic beta-cell (the sole source of circulating insulin in mice and humans) for the homologous Vps13c gene have near-normal blood sugar levels (i.e. they are "normoglycemic") and display no abnormalities in insulin secretion, making this a less likely candidate of the three to confer disease risk. By contrast, silencing of C2cd4a in pancreatic MIN6 beta-cells (which display many lf the properties of primary beta-cells) inhibits glucose-stimulated insulin secretion. Furthermore, C2CD4A expression is strongly induced by high glucose in human islets: in contrast, VPS13C mRNA levels are unaffected.

At present, mouse models in which C2cd4a or C2cd4b been deleted have not been studied using the same, detailed, approaches as those deployed for Vps13c null mice. We have therefore recently begun work to examine C2cd4b null mice provided through the IMPC. The present proposal seeks to complete the examination of this locus by using mouse genetics and cellular studies to explore the roles of C2cd4a in controlling insulin secretion and glucose homeostasis, and thus to explore the possibility that it plays a role as causal gene(s) at this genomic locus. Specifically, we will study mice rendered null globally for C2cd4a using CRISPR/Cas9-medited deletion. Firstly, we will explore the ability of the mutant mice to handle a glucose load provided either by injection into the peritoneal cavity or orally (i.e. glucose tolerance tests). Studies will be performed on mice fed (1) a regular chow direct (2) high fat, or (3) high fat/high sucrose diets. The latter resemble western diets in the human population, and lead to obesity and defective insulin secretion in man. In parallel studies we will measure beta cell mass to see whether deletion of either gene impacts the proliferation or survival of these cells. We will also measure insulin secretion into the bloodstream and the processing of the prohormone (proinsulin) to mature insulin - a process which becomes defective in human T2D. Finally, we will perform detailed studies using isolated islets from wild-type and mutant mice of "glucose sensing" by the pancreatic beta-cells. These investigations will involve advanced microscopy approaches and will determine the action of the sugar to stimulate energy metabolism, Ca2+ influx, beta cell-beta cell coordination and the release of stored insulin through regulated exocytosis.

These studies will thus provide a deep glycemic phenotyping which goes considerably beyond that undertake through the IMPC pipeline.

The majority of the work proposed will be performed by a PhD student, Ms Neda Mousavy, supported by a Diabetes UK Studentship to work on this locus. Her existing funding does not, however, cover the costs of obtaining, maintaining or characterising C2Cd4a mice.

Technical Summary

C2CD4A lies in a genomic locus associated with increased risk of Type 2 diabetes (T2D) through genome-wide association studies (GWAS). Our own (see below) and others' expression quantitative trait locus (eQTL) analysis have revealed an association between genotype at one of the previously-identified single nucleotide polymorphisms (SNPs) at this locus (rs4502156), as well as with the likely causal SNP rs7163757 (r2=0.939, D'=0.979 with rs4502156) with C2CD4A mRNA (p=0.011, n=40) levels.

Here, we will use functional genomics to explore the possibility that C2CD4A plays a role as a causal gene at this locus. Firstly, we will perform oral and intraperitoneal glucose tolerance tests in control and in mutant mice (8-20 weeks of age) deleted globally for C2cd4a using CRISPR/Cas9. Studies will subsequently be performed on mice fed (1) regular chow (2) high fat, or (3) high fat/high sucrose (60% calories) diets. Beta cell mass will be determined using optical projection tomography (OPT) alongside measurement of proliferation (ki67 staining) and apoptosis (TUNEL assay; transmission electron microscopy). Proinsulin processing will be examined in vivo (plasma proinsulin:insulin ratio) and in vitro from isolated islets. Advanced microscopy approaches will be deployed in isolated islets and beta cells to monitor glucose-stimulated energy metabolism (ATP/ADP measurements using the adenovirally-expressed recombinant probe, Perceval). Ca2+ dynamics will be examined using the entrapped fluorescent probe Fluo2, and hub-follower behaviour within the islet explored using rapid confocal analysis. The kinetics of the release of stored insulin by exocytosis will be explored using total internal reflection of fluorescence (TIRF). Depending on our findings, transcriptomic analysis (RNASeq) will also be deployed to explore changes in gene expression after C2cd4a deletion.

These studies will thus help to assess the contribution of C2CD4A to T2D risk, and its mechanisms of action.


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