Adenylate cyclase(AC) synthesizes the second messenger molecule, cyclic adenosine monophosphate (cAMP). In the insulin‐secreting β‐cell of the pancreatic islet, cAMP has critical roles in glucose‐stimulated insulin secretion and β‐cell replication. The G protein α‐subunit, Gαz, has partial tonic inhibitory activity towards AC. Our previous work has confirmed Gαz‐null mice secrete more insulin and are resistant to glucose intolerance via a significant increase in b‐cell mass. Gαz an also be activated by E‐prostanoid receptor 3 (EP3), a GPCR for the arachidonic acid metabolite, PGE2. Islets from diabetic mice and humans produce more PGE2 and express more EP3 than those from WT controls, while a EP3 antagonist restores insulin secretion. To determine the mechanisms behind the negative regulation of b‐cell function and replication by Gaz, we developed a β‐cell‐specific, cAMP biosensor expressed in intact islets, confirming, for the first time, an inverse relationship between β‐cell cAMP and Ca2+ oscillations. Loss of Gαz has the expected effects on basal β‐cell cAMP levels, and in response to an agonist of the cAMP‐stimulatory glucagon‐like peptide 1 (GLP‐1) receptor. Consistent with a lack of EP3 expression in islets from lean mice, neither agonists nor antagonists of the EP3 receptor have any effects on β‐cell cAMP, Ca2+, or the coordination between the two, regardless of Gαz expression. C57BL/6J Lepob(B6‐Ob) mice are a model of β‐cell compensation. We have shown the EP3:Gαz pathway is up‐regulated in B6‐Ob islets; raising the question as to whether up‐regulation of the this pathway is dysfunctional or beneficial. Here, we confirm B6‐Ob cAMP production and Ca2+ oscillations are up‐regulated as compared to WT mice. In addition, the anti‐phase relationship between cAMP and Ca2+ is lost as a result of reduce phosphodiesterase 1A expression. These results are also consistent with saturation of Ca2+‐regulatable AC isozymes. The EP3 agonist, sulprostone, had no effect on cAMP levels, but blocked Ca2+ influx, suggesting divergent signaling mechanisms between tonically‐active Gαz and receptor‐activated Gαz. The ability of activated Gαz to bind to Rap1GAP, sequestering it from AC, yields a hypothesis for this signaling divergence. Our current model is that cAMP signaling up‐regulated in the compensating β‐cell, ensures Rap1GAP is inactive towards Rap1, allowing Rap1 to promote cAMP‐mediated effects on exocytosis and β‐cell replication while serving as a sink for activated Gαz, inhibiting its action on AC isoforms critical for β‐cell function and mass.Support or Funding InformationI01 BX003700‐01A1, VA BLR&D; R01 DK102598, NIH/NIDDK; and ADA Innovative Basic Science Award 1‐14‐BS‐115This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.