ATP release from β cell secretory vesicles during insulin exocytosis is postulated to modulate islet hormone secretion via purinergic signaling in an autocrine or paracrine manner. Ecto-enzymes, including a family of ectonucleotidases (NTPDase) , control the concentration of extracellular nucleotides. One member of this family, ectonucleotidase triphosphate diphosphohydrolase 3 (NTPDase3) , is specifically expressed in human β cells during postnatal maturation, and its enzymatic activity is present in isolated adult human islets. The role of NTPDase3 in islet hormone secretion is not understood. We hypothesized that NTPDase3 regulates islet hormone secretion by modulating local extracellular nucleotide levels that signal through purinergic receptors expressed by islet cells. To define the cellular distribution of purinergic ecto-enzymes and nucleotide receptors in human islets, we used single-cell RNA sequencing (scRNA-seq) data. In addition to β cell NTPDase3 expression, ecto-enzymes ENPP1, and ENPP2 were abundant in α and ε cells while ecto-5'-NT and ADA were expressed in endothelial and stellate cells. These ecto-enzymes can further hydrolyze β cell-derived ATP to ADP, AMP, and adenosine which all bind purinergic receptors. We found that the ATP receptor, P2RY1, was enriched in β cells, and an adenosine receptor, ADORA2A, was expressed in α cells by both bulk and scRNA-seq. To study the function of this purinergic niche, we further assessed extracellular ATP levels which increased concomitantly (4.2E-12 vs. 4.3E-13 moles/100 IEQ/hour; n=9; p=0.03) with insulin secretion (21 vs. 60 ng/100 IEQ/hour; n=9; p=0.02) upon stimulation with 16.7 mM glucose + IBMX. To directly test NTPDase3 loss of function, we genetically manipulated human pseudoislets using an shRNA knockdown approach, resulting in a 1.7-fold increase of insulin secretion (AUC=3 vs. 5 ng/100 IEQs; n=3) . Overall, these studies provide an insight into how extracellular nucleotides and purinergic signaling mechanisms fine-tune human islet hormone secretion.
T. M. Richardson: None. D. C. Saunders: None. K. C. Coate: None. S. C. Robson: Advisory Panel; eGenesis, Consultant; Puretech, Synlogic, Research Support; Tizona Inc, Stock/Shareholder; EPurines, Purinomia. A. C. Powers: None. M. Brissova: None. C. Dai: None. R. Aramandla: None. H. H. Durai: None. C. Reihsmann: None. C. Davis: None. S. Mei: None. D. Gibson: None. S. Shrestha: None.