169-OR: LC3+ Extracellular Vesicles (EVs) Are Associated with Altered ß-Cell Autophagy, Islet Inflammation, and Diabetes

Introduction: EVs, nanoparticles housing molecular cargo, change based on parent cell health and contribute to cell-cell communication. Yet, mechanisms governing changes and physiological significance of β cell EV cargo remain unclear. Autophagy is a critical intracellular degradation and recycling process to clear damaged cellular components. EV-autophagy crosstalk is an emerging area of interest with implications for both physiological and pathological states. We have described altered β cell autophagy associated with islet inflammation and type 1 diabetes (T1D). Based on this and reports of autophagy machinery such as LC3 in EVs from other systems, we hypothesized that changes in the LC3+ EV population could yield insight into β cell autophagy. Methods: Small EVs (200nm) were isolated from INS-1 and EndoC β cells, islets, or plasma using tetraspanin (CD9, CD81, CD63) or LC3 antibody-based bead pulldown and quantified with flow cytometry. Results: We detected LC3 on the surface of EVs from β cells and normal islets. EV LC3 staining was also significantly increased by 24-hr treatment with 10 ng/mL IL-1β or 10μM caffeic acid phenethyl ester, a neutral sphingomyelinase-2 activator linked to regulation of EV subpopulations during inflammatory stress. Alternatively, LC3 staining was reduced in islet EVs from mice with β cell deletion of the critical autophagy enzyme ATG7, linking LC3+ EVs to autophagosome formation. LC3 pulldown specifically isolated β cell EVs and demonstrated the presence of proinsulin in LC3+ EVs. Finally, LC3 staining was significantly increased in plasma EVs of 20 children with new-onset T1D compared to age, sex, and BMI matched controls. Conclusion: LC3 is present on the surface of β cell EV membranes, and both β cell and circulating EV LC3 are impacted by defects in autophagy, islet inflammation, and T1D. Future studies will determine whether and how the population of LC3+ EVs participate in specific stages of autophagy or β cell pathophysiology. Disclosure J. Xu: None. M. Austin: None. A.K. Linnemann: None. E.K. Sims: Consultant; Sanofi. Board Member; American Diabetes Association. Other Relationship; American Diabetes Association, Medscape. Consultant; DRI.