Altered Extracellular Vesicle Concentration, Cargo, and Function in Diabetes

Abstract

Type 2 diabetes is a chronic age-associated degenerative metabolic disease that reflects relative insulin deficiency and resistance. Extracellular vesicles (EVs) (exosomes, microvesicles, and apoptotic bodies) are small (30-400 nm) lipid-bound vesicles capable of shuttling functional proteins, nucleic acids, and lipids as part of intercellular communication systems. Recent studies in mouse models and in cell culture suggest that EVs may modulate insulin signaling. Here, we designed cross-sectional and longitudinal cohorts of euglycemic participants and participants with prediabetes or diabetes. Individuals with diabetes had significantly higher levels of EVs in their circulation than euglycemic control participants. Using a cell-specific EV assay, we identified that levels of erythrocyte-derived EVs are higher with diabetes. We found that insulin resistance increases EV secretion. Furthermore, the levels of insulin signaling proteins were altered in EVs from individuals with high levels of insulin resistance and β-cell dysfunction. Moreover, EVs from individuals with diabetes were preferentially internalized by circulating leukocytes. Cytokine levels in the media and in EVs were higher from monocytes incubated with diabetic EVs. Microarray of these leukocytes revealed altered gene expression pathways related to cell survival, oxidative stress, and immune function. Collectively, these results suggest that insulin resistance increases the secretion of EVs, which are preferentially internalized by leukocytes, and alters leukocyte function.

Figure 1

Cohort design. A and B: Cross-sectional cohorts 1 and 2 of euglycemic individuals and individuals with diabetes. C: Longitudinal cohort design of euglycemic individuals and individuals with prediabetes or diabetes.

Figure 2

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.

Figure 3

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.

Figure 4

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.

Figure 5

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.

Figure 6

EVs from individuals with diabetes alter gene expression in monocytes. A: Plasma EVs were pooled (4.5 × 10¹¹) from several individuals from the longitudinal cohort at time 2 and grouped as either diabetic (DM) (n = 2) or euglycemic (EU) (n = 3). Cells not treated with EVs were used as another control (UNT). EVs were incubated with PBMCs for 24 h, and monocytes were isolated. Gene expression was assessed by microarray, and Gene Ontology analysis was performed. Heat map shows significant pathways (P < 0.01) related to apoptosis, immune response, oxidative stress, and vesicle formation. B: Top 20 significant downregulated and upregulated genes from the pathways in A are shown. A P value cutoff of <0.05 was used for significance. Labeling represents control group vs. treatment group. C and D: After incubation with euglycemic or diabetic EVs from cross-sectional cohort 2 (n = 3/group), total RNA and media were collected from the monocytes. Gene-specific primers were used for RT-qPCR analysis of genes from the microarray (C). D: EVs were isolated from the media using ultracentrifugation, lysed, and run along with EV-depleted media on a cytokine panel. Histograms represent the mean (SEM). *P ≤ 0.05, **P < 0.01, ***P < 0.001 by Student t test.