Altered Extracellular Vesicle Concentration, Cargo, and Function in Diabetes

Higher plasma EV concentration in individuals with diabetes. A: Plasma-derived EVs isolated from six individuals (two from each longitudinal group), two EV-depleted plasma samples, and cell lysate from 3T3-L1 cells were subjected to SDS-PAGE and probed for EV-enriched proteins. B: Electron microscopy of EVs isolated from plasma exhibit expected morphology and size. Scale bar = 500 nm. C and D: EVs were isolated from the cross-sectional diabetes cohort and concentration and size distribution were analyzed using NTA. Size distribution was averaged for each group. The area under the curve in C is shown in D. P < 0.022 by linear mixed-model regression. E: EV concentration for cross-sectional cohort 2. P < 0.016 between white euglycemic individuals (Eu) and individuals with diabetes (DM) by linear mixed-model regression. AA, African American. F: EV concentration in a longitudinal cohort showed a significant difference between the euglycemic→euglycemic and prediabetes→diabetes groups (P = 0.01) (DM, individuals with diabetes; PreDM, individuals with prediabetes). The histogram in D and line in F represent the predicted value from linear mixed-model regression. P value was determined by linear mixed-model regression on log-transformed values. G: NTA analysis of plasma EVs isolated from euglycemic individuals (n = 6) and individuals with diabetes (n = 6) from cross-sectional cohort 1 using differential ultracentrifugation. EVs were isolated from both the 10,000g and 120,000g fractions as indicated (**P < 0.01). H: Antibodies against the cell-specific markers were used to capture intact euglycemic and diabetic (n = 22/group) PKH-labeled EVs from the cross-sectional cohort 2. Fluorescent intensity was measured, and log-transformed values are shown. Dashed white line indicates average IgG signal (n = 3) for each assay. *P < 0.05 by Student t test.

Insulin signaling proteins are present in EVs and are affected by diabetes status. A: EV protein levels of the leptin receptor and phosphorylated (p)IR were measured using ELISAs, and the lines represent the predicted values from linear mixed-model regression. P = 0.01 for leptin receptor and P = 0.051 for phospho-IR for euglycemia→euglycemia group (n = 19) compared with the euglycemia→diabetes (n = 19) group (DM, diabetes; Eu, euglycemic; PreDM, prediabetes). B: EV protein levels and concentration were quantified in the longitudinal cohort at both times (euglycemia→euglycemia, n = 19, and euglycemia→diabetes, n = 19). A cross-sectional analysis was performed using time 2 (euglycemia = 19 and diabetes = 39). The relationship with HOMA-B and HOMA-IR was analyzed using linear mixed-model regression. Significant changes are indicated, and the direction of change is indicated by the up and down arrows.

Prolonged exposure to insulin impairs insulin signaling in primary cortical neurons and increases EV secretion. Neurons were untreated, pretreated with insulin (Ins.) (200 nmol/L) for 48 h, and then either untreated or treated for 30 min with insulin (200 nmol/L) or treated only for 30 min with insulin as indicated. A: Neurons were lysed and immunoblotted with anti–phosphorylated (p)AKT, total AKT, or actin antibodies. B: The conditioned media was collected from treated neurons, and the EVs were isolated by differential ultracentrifugation. EVs were lysed and analyzed by SDS-PAGE along with cell lysate from primary cortical neurons. Samples were probed using antibodies for positive and negative EV markers. C and D: NTA was used to analyze the size distribution and concentration of EVs isolated from conditioned media from treated and untreated neurons (n = 4). E: Neurons treated as described above were also incubated with 200 nmol/L wortmannin. Cell lysates were collected and immunoblotted with an anti-LC3 antibody. Arrow indicates LC3 II, and numbers indicate the ratio of LCII to LCI. Actin was used as a protein loading control. F: EVs were isolated from the conditioned media, and the concentration was measured using NTA (n = 3). All histograms represent the mean (SEM). **P < 0.01, *P < 0.05 by Student t test.

EVs from individuals with diabetes are preferentially internalized by circulating leukocytes. A: Plasma EVs (6 × 10⁸) from individuals with diabetes (n = 39) and euglycemic individuals (n = 19) from the longitudinal cohort at time 2 were incubated with PBMCs (∼200,000 cells/well) for 24 h. Cells positive for EV internalization (PKH⁺) were sorted into B cells (CD19⁺PKH⁺). B: Classical monocytes (CD14⁺⁺CD16⁻PKH⁺). C: Nonclassical monocytes (CD14⁻CD16⁺PKH⁺). D: Intermediate monocytes (CD14⁺⁺CD16⁺PKH⁺). The histograms represent means (SEM). Statistical significance was assessed by linear mixed-model regression on the log-transformed values to account for skewness of the data. DM, diabetes; Eu, euglycemic. ***P < 0.001.