PROJECT SUMMARY/ABSTRACT Diabetes mellitus affects nearly one in four Veterans and over 463 million individuals worldwide. Inadequate insulin secretion from the pancreatic β cell plays a primary role in diabetes pathogenesis; however, the mechanisms responsible for β cell failure in diabetes remain poorly understood. The long-term goal of our VA- funded research program is to identify how altered β cell calcium (Ca2+) homeostasis contributes to diabetes pathophysiology. During the last funding cycle, we identified a previously unappreciated role for impaired store- operated calcium entry (SOCE) in human and rodent models of type 2 diabetes. SOCE is a process that is activated in response to endoplasmic reticulum (ER) Ca2+ depletion and is initiated when Ca2+ dissociates from the ER Ca2+ sensor, STIM1, leading to STIM1 oligomerization and translocation to the plasma membrane. Here, STIM1 complexes with the Ca2+ selective ion channel, Orai1, leading to Orai1 opening, Ca2+ influx from the extracellular space, and subsequent transfer of Ca2+ into the ER. To study the in vivo role of SOCE, mice with β cell specific loss of STIM1 (STIM1Δβ) were generated and challenged with a high fat diet to model type 2 diabetes. Our results revealed an unusual sexually dimorphic phenotype, where female (but not male) STIM1Δβ mice displayed significantly impaired glucose tolerance as well as reduced insulin secretion and β cell mass that was associated with reduced expression of key β cell identity markers and a reciprocal increase in α cell mass. Unbiased as well as targeted transcriptional analysis revealed a reduction in G-protein coupled estrogen receptor (GPER) expression that was linked with reduced estradiol and GPER-mediated signaling. This finding was notable because loss of endogenous estradiol signaling has been linked with reduced β cell function and increased diabetes susceptibility, while estradiol has beneficial effects in both male and female β cells. Against this background, we hypothesize that impaired SOCE leads to reduced estradiol-mediated signaling through GPER, resulting in β cell de-differentiation and reduced β cell function in type 2 diabetes. To test this hypothesis, two Specific Aims are proposed in this VA Merit renewal application. In Aim 1, lineage tracing experiments will be performed to track the fate of β cells in female STIM1Δβ mice fed a high fat diet, allowing us to define whether there is bona fide β cell de-differentiation in this model. We will test whether mice with a β cell specific deletion of Orai1 (Orai1Δβ) exhibit a similar sexually dimorphic phenotype and loss of β cell identity. Finally, we will determine whether naturally occurring loss of function mutations in STIM1 and Orai1 impact directed differentiation of induced pluripotent stem cells into β cells. In Aim 2, experiments will be performed to determine how GPER and SOCE interact to regulate β cell function. Here, an ovariectomy model will be leveraged to test whet...