PROJECT SUMMARY/ABSTRACT Type 2 diabetes (T2D) is the result of an increased demand for insulin along with an inability of pancreatic β- cells to meet this demand by secreting sufficient amounts of insulin. The majority of gene loci identified by human genetic studies of T2D affect β-cell development, mass, and/or function. Yet, there is a great deal that is unknown about how β-cells secrete insulin in response to nutrient stimuli. We carried out a genetic screen of insulin secretion in 233,447 pancreatic islets ex vivo that were isolated from 483 Diversity Outbred (DO) mice. The DO mice were derived from 8 inbred mouse strains representing ~80% of the genetic diversity of all mouse strains. They have as much genetic diversity as the entire human population; ~40 million single nucleotide polymorphisms. The DO mice have been maintained for >30 generations, accumulating enough meiotic recombinations to enable high-resolution mapping of pathophysiological phenotypes. Our genetic screen identified 30 gene loci that affect insulin secretion or content of insulin or glucagon. Using our pipeline for gene identification, we chose two genes for further study, Hunk and Zfp148. We derived a whole-body knockout of Hunk and a β-cell-specific knockout of Zfp148. Islets from both of these knockout mice secreted more insulin in response to nutrient stimulation than control mice. Hunk is a protein kinase, thus we will identify the substrates of Hunk to discover how it regulates insulin secretion. Zfp148 is a transcription factor. Thus, we will identify the direct transcriptional targets of Zfp148. We will apply our bioinformatic pipeline to identify additional genes from the 30 gene loci identified in our screen and provide them to the research community. Functional studies will be performed in genetically edited mice, genetically edited human ES-derived β-cells, and human islets. These studies will discover novel genes and pathways involved in β-cell function and T2D susceptibility.