Role of Extracellular Vesicle: Autophagy Pathway Crosstalk in Type 2 Diabetes Pathogenesis - PROJECT SUMMARY/ABSTRACT The prevalence of Type 2 diabetes (T2D) is an expanding global issue that continues to impact several developing nations. The onset of pancreatic β-cell functional failure is a central feature to the pathophysiology of T2D and is mediated in part by diabetogenic stressors including chronically elevated free fatty acids (lipotoxicity), low-grade systemic inflammation, and proteotoxicity. Due to their high protein synthetic burden, β- cells employ autophagy and endo/lysosomal pathways to reduce protein aggregates and protect β-cell mass and function. Recently, two endomembrane systems – small extracellular vesicles (sEVs; 30-150 nm sized nanoparticles) and autophagy have been shown to work in tandem to facilitate cellular adaptation and intercellular communication. Under diabetogenic stress, β-cell defects in autophagy mediated by auto/lysosomal dysfunction have been shown to occur, while conversely our group and others have noted enhanced sEV secretion in these conditions. However, what remains elusive is our understanding of the molecular mechanisms underlying the dis-balance between autophagy and sEV secretory pathways and potential therapeutic strategies to re-balance both in order to improve functional β-cell mass. Thus, our central hypothesis is that diabetogenic stress-mediated β-cell lysosomal dysfunction occurs in part, through activation of a novel secretory autophagy pathway to enhance β-cell sEV secretion. In Specific Aim 1, we will use in vitro and ex vivo, mouse and human diabetic models to investigate the molecular mechanisms associated with diabetogenic stress-mediated enhanced β-cell sEV secretion through activation of a novel sEV-based secretory autophagy pathway. We will use state-of-the-art EV based tools and technologies to visualize and quantify events that will determine activation of secretory autophagy. Furthermore, we will use sophisticated cell biology techniques to delineate the mechanism of preferential β-cell sEV release mediated by diabetogenic stress-induced amphisome de- acidification events. Our preliminary data reveal significant improvements in β-cell function and a reduction in β- cell sEV concentration under diabetogenic stress with pharmacological agents to either repress sEV biogenesis/release or enhance autophagy. Thus, in Specific Aim 2, we will explore in vivo and in vitro pharmacological, nutritional, and genetic therapeutic approaches to rebalance sEV secretory:autophagy pathway flux under diabetogenic conditions with the goal of improving functional β-cell mass. Using these approaches, we will first test the impact of a) inhibition of sEV biogenesis/release; b) enhancing autophagy; c) using a combinatorial approach to determine alterations in sEV release, autophagy induction, and β-cell function. Taken together, completion of the proposed application will provide novel insight into the molecular mechanisms that regulate sEV biogenesis and autophagy crosstalk during β-cell functional failure in T2D. Moreover, these studies will uncover new potential therapeutic approaches to restore the balance between both pathways with the goal to preserve and/or restore functional β-cell mass.
