Proinflammatory Stress Activates Neutral Sphingomyelinase 2–Based Generation of a Ceramide-Enriched β-Cell EV Subpopulation

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 small 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 abundance, as well as small EV ceramide species. Direct chemical activation or genetic knockdown of nSMase2, chemical treatment to inhibit cell death pathways, or treatment with a glucagon-like peptide-1 (GLP-1) receptor agonist also modulated β-cell EV ceramide. RNA sequencing of ceramide-enriched EVs identified a distinct set of miRNAs linked to β-cell function and identity. EV treatment from cytokine-exposed parent cells inhibited peak glucose-stimulated insulin secretion in wild-type recipient cells; this effect was abrogated when using EVs from nSMase2 knockdown parent cells. Finally, plasma EVs in children with recent-onset type 1 diabetes showed increases in multiple ceramide species. 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 and death. ARTICLE HIGHLIGHTS Mechanisms connecting β-cell stress to extracellular vesicle (EV) cargo and diabetes are poorly defined. Does β-cell inflammatory stress engage neutral sphingomyelinase 2 (nSMase2)–dependent EV formation to generate ceramide-enriched small EVs? Proinflammatory cytokines increased β-cell small EV ceramide via increases in nSMase2. Ceramide-enriched EVs housed distinct cargo linked to insulin signaling, and ceramide species were enriched in plasma EVs from individuals with type 1 diabetes. Ceramide-enriched EV populations are a potential contributor to β-cell EV-related paracrine signaling.