Type 2 diabetes risk variant effects on mitochondrial (patho)physiology - PROJECT SUMMARY / ABSTRACT Type 2 diabetes (T2D) results when pancreatic islet β-cells fail to secrete sufficient insulin to meet peripheral insulin demand. Mitochondrial bioenergetics is central to the (patho)physiology of β-cell (dys)function, and recent work suggests that β-cell mitochondrial dysfunction precedes the development of T2D in β-cells from donors with impaired glucose tolerance (or pre-diabetes). Mitochondrial defects have been reported in the β-cells of human T2D patients, but the etiology of mitochondrial dysfunction in T2D is unknown. Such mechanistic knowledge is necessary to guide strategies to prevent or treat islet failure and T2D. Importantly, genome-wide association studies (GWAS) link single nucleotide polymorphisms (SNPs) in >500 genetic loci to T2D and islet dysfunction-related metabolic traits. The majority of these SNPs are non-coding and overlap regulatory elements (REs) with broad transcriptional implications for affected cells. In this study, we combine our expertise in the genomics of T2D, (epi)genomic modification, and mitochondrial function in β-cells to bridge the gap from genomic association to mechanistic understanding. We hypothesize that non-coding T2D SNPs cause β-cell dysfunction by altering RE use or activity, thereby changing expression of effector genes that directly impair mitochondrial health. To test this, we propose to use sophisticated (epi)genomic editing tools in human islets and β-cell specific mouse models for physiological relevance and validation in two complementary Aims. In Aim 1, we will test RE– effector gene links in human islets using CRISPR-QTL. In parallel, we will assess T2D risk allele effects on RE chromatin accessibility, activity, transcription factor binding, and β-cell expression of putative mitochondrial T2D effector genes using complementary in vivo (single cell chromatin accessibility, histone acetylation, and expression quantitative trait locus analysis of primary human islets) and in vitro (reporter gene, DNA-binding assay) approaches. Finally, we will determine the consequences of effector gene perturbation on mitochondrial phenotypes, β-cell viability, and insulin content and secretion in human islets and EndoC-βH3 cells. In Aim 2, we harness β-cell-specific knockout mouse models to assign function to two high-priority mitochondrial T2D effector genes in glycemic control, β-cell mass/function, and mitochondrial metabolism. Further, we will address the importance of these mitochondrial T2D effector genes for β-cell compensation to peripheral insulin resistance following diet-induced obesity. Finally, we will use (epi)genomic editing tools in human islets to determine if mitochondrial T2D effector genes impair β-cell function and glycemic control in ex vivo assays as well as after islet transplantation into immunodeficient mice. Completion of this study will generate new variant-to-function connections that assign molecular and cellular functions to T2D risk alleles, identify novel therapeutic targets, and provide important knowledge to guide subsequent strategies to prevent or treat β-cell failure and T2D.
