Pancreatic ß-cells store insulin and release it in response to an elevation of blood glucose. Additionally, with each meal, healthy ß-cells synthesize new insulin to replace what they have lost. The synthesis pathway begins with introducing into the endoplasmic reticulum (ER) on average ~6,000 new proinsulin molecules every second per ß-cell. Under normal conditions, these molecules must fold their insulin moiety to the native state, including the formation of 3 evolutionarily conserved disulfide bonds. However, we have now definitively identified proinsulin misfolded states that occur in the ER of human islets, rodent islets, or ß-cell lines, and get worse (more abundant) with perturbations of the ER folding environment. We believe that this is an important contributor to abnormally increased ß-cell ER stress – and it is even worse in the disease of Mutant INS-gene induced Diabetes of Youth (MIDY) – caused by any one of >30 heterozygous (dominant-negative) INS gene coding sequence mutations. A portion of the work in the current proposal will focus on MIDY models, but we also present important new data highlighting the formation of misfolded protein complexes containing proinsulin that are especially abundant in islet ß-cells without INS mutation, preceding the onset of type 2 diabetes (T2D). In our first Aim, we will further analyze the ER environment and diabetogenic conditions promoting the formation of misfolded complexes of proinsulin, using molecular, cellular, and physiological tools. In our second Aim, we propose to understand more about the molecular mechanism of misfolding by using various MIDY models, and particularly, we propose experiments that will allow us to determine how much misfolded proinsulin must accumulate in order to trigger diabetes. Finally, in our third Aim, we propose a strategy to limit proinsulin misfolding with the intent to ameliorate, delay, or prevent diabetes.