Proinflammatory stress activates neutral sphingomyelinase 2 based generation of a ceramide-enriched β cell EV subpopulation

{beta} cell extracellular vesicles (EVs) play a role as paracrine effectors in islet health, yet mechanisms connecting {beta} cell stress to changes in EV cargo and potential impacts on diabetes remain poorly defined. We hypothesized that {beta} cell inflammatory stress engages neutral sphingomyelinase 2 (nSMase2)-dependent EV formation pathways, generating ceramide-enriched EVs that could impact surrounding {beta} cells. Consistent with this, proinflammatory cytokine treatment of INS-1 {beta} cells and human islets concurrently increased {beta} cell nSMase2 and ceramide expression, as well as EV ceramide staining. Direct chemical activation or genetic knockdown of nSMase2, or treatment with a GLP-1 receptor agonist also modulated cellular and EV ceramide. Small RNA sequencing of ceramide-enriched EVs identified a distinct set of miRNAs linked to {beta} cell function and identity. Coculture experiments using CD9-GFP tagged INS-1 cell EVs demonstrated that either cytokine treatment or chemical nSMase2 activation increased EV transfer to recipient cells. Children with recent-onset T1D showed no abnormalities in circulating ceramide-enriched EVs, suggesting a localized, rather than systemic phenomenon. These findings highlight nSMase2 as a regulator of {beta} cell EV cargo and identify ceramide-enriched EV populations as a contributor to EV-related paracrine signaling under conditions of {beta} cell inflammatory stress. Article HighlightsO_ST_ABSa. Why did we undertake this study?C_ST_ABSMechanisms connecting {beta} cell stress to changes in extracellular vesicle (EV) cargo and potential impacts on diabetes are poorly defined. b. What is the specific question we wanted to answer?Does {beta} cell inflammatory stress engage neutral sphingomyelinase 2 (nSMase2)-dependent EV formation pathways to generate ceramide-enriched EVs. c. What did we find?Proinflammatory cytokine treatment of {beta} cells increased {beta} cell ceramide expression, along with EV ceramide in part via increases in nSMase2. Ceramide-enriched EVs housed a distinct set of miRNAs linked to insulin signaling. Both cytokine treatment and nSMase2 activation increase EV transfer to other {beta} cells. d. What are the implications of our findings?Our findings highlight nSMase2 as a regulator of {beta} cell EV cargo and identify ceramide-enriched EV populations as a contributor to EV-related paracrine signaling under conditions of {beta} cell inflammatory stress.