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Increased inflammation renders the vascular endothelium susceptible to atherosclerosis and thrombosis. Insulin has been shown to influence vascular function. Hyperinsulinemia is associated with a proinflammatory endothelial phenotype. In vitro, insulin has been shown to increase the inflammatory transcription factor NF-kB. Extracellular vesicles, particularly endothelial cell-derived microvesicles (EMVs), have emerged as mechanistic factors mediating of endothelial inflammation and, in turn, vascular disease. Circulating EMVs are elevated in metabolic conditions associated with hyperinsulinemia. However, whether insulin stimulates EMV release is unknown, and, if so, whether insulin-derived EMVs are proinflammatory. The experimental aims of this study were to determine: 1) if insulin stimulates EMV release from endothelial cells; and 2) whether insulin-derived EMVs promote a proinflammatory endothelial phenotype. Human umbilical vein endothelial cells (HUVECs) were cultured (3rd passage) and incubated with 1 nM insulin (concentration representing a hyperinsulinemic state) for 24 h. EMVs released into the supernatant from cells treated with insulin or vehicle were isolated and quantified by flow cytometry (CD144-PE). In separate experiments, HUVECs (2x106 cells/condition) were treated with either insulin, insulin-induced EMVs or control EMVs for 24 h. EMV release was markedly higher (~185%; P<0.05) in cells treated with insulin compared with control (88±10 vs. 31±5 EMV/μL). Insulin-generated EMVs induced significantly higher release of interleukin (IL)-6 (25.4±1.5 vs 20.1±1.2 pg/mL) and IL-8 (37.8±2.5 vs 27.1±2.7 pg/mL) than control EMVs. Although total NF-kB p65 expression was not significantly altered; insulin-derived EMVs induced ~30% higher (P<0.05) expression of phosphorylated-NF-kB p65 (Ser536; active NF-kB) (8.5±0.4 vs. 6.5±0.3 AU). The adverse effects of insulin-generated EMVs on IL-6, IL-8, and pNF-kB p65 were consistent with the proinflammatory effects of insulin. In summary, insulin induces an EMV phenotype that adversely affects endothelial cell inflammation. Insulin-generated EMVs may contribute to the proinflammatory vascular effects of insulin. Nothing to disclose This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.