Abstract Glucose metabolism generates important signals that regulate several facets of β-cell health, including insulin secretion, proliferation, survival, and differentiation. We have found that the TCA cycle enzyme pyruvate carboxylase (PC), which promotes metabolic signals that regulate insulin secretion, has previously unappreciated roles in β-cell stress mitigation by promoting urea cycle activity and de novo glutathione (GSH) synthesis. These PC-directed pathways can promote a dual defense mechanism, whereby nitric oxide (NO) synthesis from arginine is reduced as arginine is utilized for ureagenesis and antioxidant capacity is increased by de novo GSH synthesis in the face of diabetes-related stress stimuli. Both pathways receive input from glucose through PC activity and are modulated by phosphorylation of BAD, a GK binding and activating protein that drives a broad β-cell protective program. This renewal application addresses key questions that stem from these findings: What are the molecular mechanistic components of this PC-driven β-cell protective program and how do they contribute to β- cell mass dynamics? We will address these questions in two aims. First, we will dissect the downstream effectors of the urea cycle pathway that are required for β-cell protection. Our prediction is that beyond changing the balance between urea and NO production, urea cycle activation provides biosynthetic intermediates that promote growth, including ornithine and polyamines. We will also examine how glucose is connected to these pathways using metabolomics and metabolic tracing studies combined with genetic and pharmacologic perturbations of these pathways. Similarly, we will examine the route for glucose to GSH synthesis and how this antioxidant mechanism factors into PC-mediated protection. We will then test the relevance of these pathways in protecting donor islets in a marginal mass islet transplantation model (Aim 1). Second, we will test the contributions of these pathways to β-cell mass adaptation in response to high fat diet (HFD) using two complementary approaches; a β-cell-specific PC knockout mouse model treated with HFD and assessing the response of donor islet grafts to (HFD) treatment of transplanted mice (Aim 2). These studies will provide an integrated picture of the pathways connecting phospho-BAD, GK and PC activation to β-cell stress response and adaptation. In the fullness of time, understanding these connections will yield valuable insights into the most effective strategies to capture and mimic the protective aspects of glucose signaling for preservation/restoration of functional β-cell mass.