PROJECT SUMMARY/ABSTRACT Pancreatic β-cell failure in a distinctive feature of Type 2 diabetes mellitus (T2DM) and therefore preservation of β-cell health has been identified as a critical barrier for the development of successful preventative and treatment strategies in T2DM. Primary features of β-cell failure include insulin secretory dysfunction, loss of transcriptional identity/β-cell dedifferentiation, and β-cell loss, with growing evidence pointing to impaired endoplasmic reticulum (ER) proteostasis as a key driver of this process. Recent evidence suggests that environmental stress conditions that produce disruptions of daily fasting/feeding circadian cycles (i.e. circadian disruption, CD) lead to glucose intolerance, hyperglycemia, and promote β-cell failure in T2DM. However, the molecular mechanisms underlying circadian control of β-cell function and ER proteostasis remain unknown. In this regard, our preliminary studies determined that CD-mediated abrogation of normal fasting/feeding cycles is a potent physiological inducer of β- cell functional failure, and this process is molecularly mediated through loss of β-cell expression/activity of circadian transcription factor D-box binding PAR bZIP transcription factor (Dbp). Therefore, the key objective of the proposal is to test the hypothesis that Dbp is an important regulator of β-cell circadian function through rhythmic activation of transcripts regulating insulin secretion and ER proteostasis, whereas loss of Dbp expression, as occurs in β-cells in response to circadian disruption and diet-induced obesity, promotes β-cell functional decline in T2DM. To address this, Specific Aim 1 will utilize novel conditional genetic loss and gain-of- function Dbp mouse models to establish a causative relationship between circadian Dbp expression and the regulation of β-cell function, ER proteostasis, and transcriptional identity. In addition, we will also examine whether Dbp regulates β-cell function and transcription in human β-cells utilizing viral gain/loss of function techniques concurrent with transplantation of human stem cell-derived β-cells into immunodeficient mice. Specific Aim 2 will utilize β-cell-specific Dbp luciferase reporter mice and systems biology multiomics approaches (RNAseq + scATAC seq) to 1) identify molecular mechanisms by which obesity disrupts circadian regulation of Dbp expression and corresponding β-cell circadian clock function, and 2) test novel strategies designed to restore normal functionality of β-cell circadian clocks in obesity as means to prevent β-cell failure in T2DM. Taken together, successful completion of proposed studies will uncover novel molecular mechanisms through which β- cells integrate and respond to circadian changes in nutritional availability and will provide potential therapeutic targets for prevention and treatment of T2DM.