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

Abstract β cell extracellular vesicles (EVs) play a role as paracrine effectors in islet health, yet mechanisms connecting β cell stress to changes in EV cargo and potential impacts on diabetes remain poorly defined. We hypothesized that β cell inflammatory stress engages neutral sphingomyelinase 2 (nSMase2)-dependent EV formation pathways, generating ceramide-enriched EVs that could impact surrounding β cells. Consistent with this, proinflammatory cytokine treatment of INS-1 β cells and human islets concurrently increased β 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 β 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 β cell EV cargo and identify ceramide-enriched EV populations as a contributor to EV-related paracrine signaling under conditions of β cell inflammatory stress. Article Highlights a. Why did we undertake this study? Mechanisms connecting β 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 β 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 β cells increased β 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 β cells. d. What are the implications of our findings? Our findings highlight nSMase2 as a regulator of β cell EV cargo and identify ceramide-enriched EV populations as a contributor to EV-related paracrine signaling under conditions of β cell inflammatory stress.