Type 2 diabetes (T2D) is a chronic disease that affects nearly 25% of U.S. Veterans, a prevalence that exceeds that among the civilian population, and represents a growing challenge to the VA healthcare system. T2D comprises a heterogeneous set of metabolic disorders that commonly arises from obesity-associated insulin resistance, inadequate insulin production and secretion from pancreatic islet beta cells, and dysregulated (often increased) secretion of glucagon from pancreatic islet alpha cells that collectively lead to hyperglycemia. Although it has been recognized for decades that diabetes is a bi-hormonal disease reflecting pathology in both pancreatic islet beta and alpha cells, the mechanisms governing human alpha cell function in physiological or pathological settings are virtually unknown. Therefore, discovery of molecular mechanisms that control human alpha cell gene regulation and hormone secretion are urgently needed. Islet-enriched transcription factors (TF) are critical regulators of alpha and beta cell development and function, with alterations in their activity leading to diabetes. One such TF, MAFB, was shown to be compromised early on in human islets in response to diabetes-relevant cellular stressors, including oxidative stress, high fat dietinduced insulin resistance, and hyperglycemia. Furthermore, both type 1 diabetes and T2D are associated with marked downregulation of MAFB expression in human alpha and beta cells, implicating compromised MAFB activity with islet cell dysfunction. I hypothesize that MAFB is a critical regulator in human alpha cells, with its loss contributing to dysregulated g/ucagon secretion and glucose homeostasis pathophysiologically. To test this hypothesis, I will use genetic manipulation of MAFB levels in a human pseudoislet system combined with advanced transplantation, functional, and transcriptomic analyses to determine how loss of MAFB impacts human alpha cell gene regulation and function in vitro and in vivo (Aim 1 ). In addition, I will comprehensively define the functional and molecular changes that occur in transplanted human alpha cells in vivo under conditions of chronic insulin resistance, an acquired pathological state that impacts MAFB (Aim 2). A key component of my strategy is use of a new transplant-tolerant glucagon knockout mouse model (i.e., GKONSG), allowing for the first-time longitudinal examination of (patho)physiologic changes in human alpha cells and glucagon secretion in vivo in response to conditions that mimic the genetic and metabolic milieu of human T2D. These experiments should provide fundamental new insights into the physiologic and genetic mechanisms controlling human alpha cell activity in healthy and diseased pancreatic islets and may lead to new clinically actionable information to treat the underlying causes of T2D. Additionally, the structured training and mentored research of this career development award will form a strong foundation for my independent research...