In early T1D, β cells are subjected to stresses such as inflammation, putative viral infections and increased ER protein load. Under these conditions, β cells activate the integrated stress response, which is highlighted by a block in the translation of mRNAs. These translationally-inhibited mRNAs are compartmentalized into non-membranous inclusions known as stress granules (SGs). During chronic stress, SGs may be sequestered into endosomes and released as components of extracellular vesicles (EVs) into circulation. We hypothesized, the RNA content of EVs may serve as a reflection of ongoing β cell stress. To test this possibility, we measured EV-associated preproinsulin (PPI) mRNA from supernatant of a cultured human β cell line and human islets treated with and without cytokines (IL-1β and IFN-γ) to mimic T1D conditions. Cytokine treatment resulted in a block in mRNA translation initiation, decreased protein synthesis, and increased formation of SGs. Studies using single molecule FISH confirmed that in stressed human β cells there is significant colocalization of PPI mRNA with G3BP1, a marker of SGs. Moreover, stressed human β cells expressing G3BP1-RFP and CD63-GFP (a marker of EVs) exhibited co-localization of SGs with EVs, suggesting that SG contents incorporate into EVs. PPI mRNA was recoverable almost exclusively in EVs isolated from supernatants of stressed human islet cultures. Pharmacological inhibition of EV release decreased PPI mRNA content in the culture media, implying that PPI mRNA is released from β cells within EVs. Droplet digital PCR analysis demonstrated significantly higher levels of PPI mRNA in circulation of new-onset T1D subjects compared to healthy, age-matched controls. Taken together, our data demonstrate that PPI mRNA is released from stressed β cells as a consequence of translational blockade, SG formation, and EV release, and indicate that circulating PPI mRNA might serve as a biomarker reflective of β cell stress.
F. Syed: None. B.F. Maier: None. E. Anderson-Baucum: None. T.L. Mastracci: None. C. Evans-Molina: Consultant; Self; Bristol-Myers Squibb. R. Mirmira: Advisory Panel; Self; Hibercell, Sigilon Therapeutics, Veralox Therapeutics. Employee; Spouse/Partner; Eli Lilly and Company.
National Institute of Diabetes and Digestive and Kidney Diseases (UC4DK104166 to R.M., C.E-M.), (R01DK60581 to R.M.), (R01DK093954 to C.E-M.); U.S. Department of Veterans Affairs (I01BX001733 to C.E-M.); JDRF (3-SRA-2014-41-Q-R to C.E-M, R.M.), (3-PDF-2016-199-A-N to F.S.); Sigma Beta Sorority; Ball Brothers Foundation; George and Frances Ball Foundation; Holiday Management Foundation (to C.E-M., R.M.)