Final Report Summary - NEW IRE-1 ACTIVITY (Insulin/IGF-1 Signaling Regulates Novel Activities of the ER Stress Response Gene ire-1) The insulin/IGF-1 signaling pathway is a central pathway that controls many fundamental biological processes. Using a transgenic C. elegans strain harboring a GFP-labeled insulin fusion protein, we discovered that the combined inactivation of the ER stress sensor ire-1 and reducing insulin/IGF1 signaling resulted in a striking accumulation of the secreted protein reporter in the body cavity of the animals. This grant aimed to combine molecular and genetic approaches to investigate the underlying molecular mechanism of this phenomenon by addressing the following:1) What cellular mechanism accounts for the accumulation of secreted proteins in the body cavity of ire-1 mutants upon reducing insulin/IGF-1 signaling? We discovered that animals deficient in ire-1 have defective coelomocyte cells, which are essential for the clearance of secreted material from the body cavity. Concomitantly, the production and secretion of labeled secreted proteins is abrogated in ire-1-defficient animals. In fact, secretory protein metabolism is so bad in these animals that the luminal substrates remain trapped within the ER. We discovered that reducing insulin/IGF1 signaling restored the production and secretion of the labeled secreted insulin from its producing cells, but did not restore the function of the coelomocyte cells. This combination of restored secretory protein metabolism in the producing cells together with dysfunctional coelomocytes leads to the aberrant accumulation of secreted proteins in the body cavity of ire-1; daf-2 double mutants.2) What molecular mechanism may account for the restored metabolism of insulin proteins in ire-1 mutants upon reducing insulin/IGF-1 signaling? We discovered the improvement in ER homeostasis and function in these animals can be attributed, at least in part, to the facilitation of the clearance of misfolded proteins passing through the secretory pathway. 3) What genes contribute to the accumulation of secreted proteins in the body cavity of ire-1 mutants with reduced insulin/IGF-1 signaling?daf-16 encodes a transcription factor that acts downstream to the daf-2 insulin/IGF1 receptor and is required for most of its functions. We established that daf-16 is also required for the restored secretion of GFP-labeled insulin fusion protein into the body cavity of ire-1 mutants upon reducing insulin IGF1 signaling. We further identified downstream genes, whose expression is regulated by DAF-16, whose inactivation produce a similar phenotype. Analysis of the cellular and molecular activities of these genes provides an insight into the mechanism by which ERAD is improved when insulin/IGF1 signaling is reduced.In summary, this research provides novel insights into the regulation of secretory protein metabolism in general, and into the regulation of insulin metabolism specifically. This research clearly demonstrates that conditions that mimic reduced insulin-IGF-1 signaling can significantly improve secretory protein metabolism. These studies may be of particular importance and relevance in the context of diabetes, where insulin/IGF-1 signaling is dysfunctional, insulin levels are deregulated and insulin-secreting cells are over-loaded and suffer from ER stress.