Ketosis Suppression and Ageing (KetoSAge) Trial: Effect of Hypoketonaemia on Extracellular Vesicle Profiles in Healthy Premenopausal Women

Extracellular vesicles (EVs) mediate endocrine and metabolic signalling. EV responses to shifts in substrate utilisation, from euketonaemia to hypoketonaemia, remain poorly understood. Chronic hyperinsulinaemia suppresses ketogenesis, causing hypoketonaemia; however, its impact on EV profiles in metabolically healthy individuals has not been characterised. We investigated the effects of hypoketonaemia on EV profiles in the KetoSAge cohort, examining associations between EV concentration, mean size, and modal size and metabolic–endocrine biomarkers. Ten lean (BMI, 20.52 ± 1.39 kg/m2), healthy premenopausal women (mean age, 32.30 ± 8.97 years) maintaining euketonaemia (mean 3.9 ± 2.3 years) completed a three-phase crossover trial, comprising 21 days each of: euketonaemia (baseline), induced-hypoketonaemia, and return-to-euketonaemia. No statistically significant phase-level differences in EV concentration or size parameters were detected. Linear mixed-effects analyses identified nominally significant associations between EV concentration and adiponectin, free T3, bilirubin, luteinising hormone, and epidermal growth factor, between EV mean size and HDL-cholesterol and between EV modal size and insulin, HOMA-IR, beta-hydroxybutyrate, GKI, thyroid hormones, ALT, AST, IL-6 and oestrogen (all unadjusted p < 0.05). However, none of these associations remained statistically significant after false discovery rate correction (FDR < 0.10). Hypoketonaemia via insulin-compensated euglycaemia revealed coordinated EV–biomarker relationships across metabolic–endocrine, hepatic and inflammatory pathways. Although exploratory, these findings suggest that EV profiles may reflect an insulin-responsive signalling network linked to the systemic bioenergetic state, highlighting their potential as early indicators of metabolic–endocrine adaptation.