Type 2 diabetes (T2D) plagues nearly 10% of the US population; a shocking 86 million more have prediabetes, and show signs of impaired glucose tolerance. Compounding this problem, certain insulin-sensitizing drugs are undergoing global market suspensions due to severe health complications, leaving prediabetic and T2D patients with fewer treatment options. Because multi-tissue dysfunction contributes to prediabetes and progression to T2D, prevention or reversal of these diseases requires a multi-pronged approach. Specifically, pancreatic ß-cell dysfunction and skeletal muscle insulin resistance are primary features of human prediabetes and T2D. DOC2B, a key regulator of exocytosis proteins, is required for normal insulin secretion (from islet ß-cells) and peripheral insulin sensitivity for normal glucose uptake (in skeletal muscle); loss of DOC2B abundance is associated with prediabetes and T2D in human and rodent islets and skeletal muscle, and DOC2B+/- mice are more susceptible to impaired glucose tolerance. Thus, the long-term goal is to understand how vesicle exocytosis mechanisms can be manipulated to prevent and/or reverse prediabetes and halt the progression to T2D. Recent discoveries of new functions of DOC2B in exocytosis, plus new methods to target DOC2B abundance and/or capitalize on its tissue-specific functions to control glycemic dysregulation, offer an enticing and untapped opportunity for disease intervention. The objective of this application is to determine how DOC2B enrichment enhances ß-cell insulin secretion and skeletal muscle insulin sensitivity in human tissues, in vivo, and at the molecular level. Preliminary data show that DOC2B upregulation in ß-cells increases the function of human T2D islets, diminishes cytokine-induced apoptosis, and protects mice from diabetogenic stimuli. Moreover, DOC2B functions in skeletal muscle to increase vital complex formation amongst exocytosis proteins, and a peptide fragment of DOC2B can recapitulate this function. The central hypothesis is that DOC2B is essential for insulin release and glucose uptake in ß-cells and skeletal muscle, respectively, and that DOC2B upregulation can improve insulin/glucose regulation to prevent or reverse prediabetes. The rationale for the proposed research is that once it is known how DOC2B enrichment protects glucose homeostasis, DOC2B can be manipulated to prevent or reverse prediabetes. Two Specific Aims are designed to test this: 1) Evaluate DOC2B enrichment in the prevention/reversal of diabetogenic stimuli-induced ß-cell dysfunction and demise, and 2) Delineate how DOC2B enrichment in skeletal muscle promotes insulin sensitivity. We will use innovative inducible ß-cell- and skeletal muscle-specific DOC2B transgenic mice for mechanistic discovery. We will also test translational approaches for increasing DOC2B expression and function, using live-cell imaging biosensors paired with biochemical assays in human cells/tissues. The results will positively impact efforts to ameliorate prediabetes as the identified mechanisms are highly likely to provide new therapeutic strategies.