Abstract Aims/hypothesis Genome-wide studies have uncovered multiple independent signals at the RREB1 locus associated with altered type 2 diabetes risk and related glycemic traits. However, little is known about the function of the zinc finger transcription factor RREB1 in glucose homeostasis or how changes in its expression and/or function influence diabetes risk. Methods A zebrafish model lacking rreb1a and rreb1b was used to study the effect of RREB1 loss in vivo . Using transcriptomic and cellular phenotyping of a human beta cell model (EndoC-βH1) and human induced pluripotent stem cell (hiPSC)-derived beta-like cells, we investigated how loss of RREB1 expression and activity affects pancreatic endocrine cell development and function. Ex vivo measurements of human islet function were performed in donor islets from carriers of RREB1 T2D-risk alleles. Results CRISPR-Cas9-mediated loss of rreb1a and rreb1b function in zebrafish supports an in vivo role for the transcription factor in beta cell mass, beta cell insulin expression, and glucose levels. Loss of RREB1 reduced insulin gene expression and cellular insulin content in EndoC-βH1 cells, and impaired insulin secretion under prolonged stimulation. Transcriptomic analysis of RREB1 knockdown and knockout EndoC-βH1 cells supports RREB1 as a novel regulator of genes involved in insulin secretion. In vitro differentiation of RREB1 KO/KO hiPSCs revealed a dysregulation of pro-endocrine cell genes, including RFX family members, suggesting that RREB1 also regulates genes involved in endocrine cell development. Human donor islets from carriers of T2D-risk alleles in RREB1 have altered glucose-stimulated insulin secretion ex vivo , consistent with RREB1 regulating islet cell function. Conclusions/interpretation Together, our results indicate that RREB1 regulates beta cell function by transcriptionally regulating the expression of genes involved in beta cell development and function. Research in context What is already known about this subject? Human genetic variation in RREB1 is associated with altered diabetes risk, variation in glycemic, and anthropometric traits RREB1 is a transcription factor that binds to Ras-responsive elements and is expressed in multiple diabetes relevant tissues, including pancreatic islets What is the key question? How does altered expression or function of RREB1 influence diabetes risk? What are the new findings? Knockdown and knockout of RREB1 in mature human EndoC-βH1 cells reduces expression of insulin transcript and cellular content, as well as insulin secretion under prolonged stress Carriers of the T2D-risk RREB1 coding allele trend towards reduced insulin content, but have improved glucose-stimulated insulin secretion A loss-of-function zebrafish model suggests that RREB1 is required for insulin expression How might this impact on clinical practice in the foreseeable future? RREB1 controls beta cell function and whole-body glucose homeostasis by transcriptionally regulating the development and function of pancreatic beta cells

Loss of RREB1 in pancreatic beta cells reduces cellular insulin content and affects endocrine cell gene expression