Project Summary Islet glucose-stimulated insulin and somatostatin (SST) secretion are perturbed in patients with type-2 diabetes (T2D) and in animal models of the disease, which contributes to disrupted glucose homeostasis. It is generally accepted that secretagogues stimulate hormone secretion from -cells and -cells in response to elevated intra- cellular Ca2. However, the mechanisms that control inhibition of islet Ca2+ handling via Gi/o-coupled receptors (Gi/o-GPCRs) and how they are altered in T2D are largely unknown. Data from our lab finds that Gi/o-GPCRs reduce islet Ca2+ entry via Src tyrosine kinase-mediated activation of Na+/K+-ATPase (NKA), which hyperpolarizes membrane potential (Δψp) and limits insulin secretion. Further data show that Protein kinase A (PKA) activation by Gs-coupled receptors inhibits islet NKA activity and stimulates Ca2+ entry. Moreover, we find that islet SST provides paracrine signaling that slows glucose-stimulated -cell Ca2+ oscillations via oscillations in NKA activity, which depends on the action of Src tyrosine kinase and PKA. Finally, our preliminary data provide the first evidence that diabetic conditions diminish islet NKA activity, which contributes to perturbations in glucose and GPCR control of Ca2+ handling. Based on these exciting preliminary data, the overall objective of this pro- posal is to elucidate how islet NKA is controlled and becomes disrupted during the pathogenesis of diabetes. This project will test the central hypothesis that that islet NKA activation by tyrosine kinases limits Ca2+ entry and hormone secretion through Δψp hyperpolarization; whereas, PKA inhibition of islet NKAs enhances Ca2+ entry and hormone secretion by depolarizing Δψp. The rationale that underlies this project is that understanding sig- naling that integrates NKA modulation of islet cell Ca2+ handling and hormone secretion will expose novel thera- peutic targets for restoring glucose-stimulated hormone secretion in T2D. This project will be accomplished with the following two specific aims: 1) Determine the mechanisms regulating NKA control of β-cell function in health and diabetes; and 2) Determine how NKA modulates -cell function and dysfunction. Under the first aim, trans- genic mice with -cell ablation of the - - and -subunits of the NKA complex subunits as well as human -cells with knockdown of NKA - - and -subunits will be utilized to assess the roles of NKA during secretagogue and Gi/o-GPCR modulation of -cell Ca2+ handling and insulin secretion. Aim1 will also determine how diabetic con- ditions impact NKA signaling and insulin secretion. Under the second aim, NKA control of -cell Ca2+ handling and function will be determined in mice with -cell specific ablation of NKA - - and -subunits or in human pseudoislets with -cell specific knockdown of NKA - - and -subunits. Furthermore, Aim2 will determine how reduced NKA function in -cells under the stressful conditions associated with diabet...