Extended Data Fig. 3: Glucose tolerance testing in mice in response to extracellular vesicles derived from various sources and glucose-stimulated extracellular vesicle secretion.
(A-C) The GTT glucose area under the curve (AUC) in control (A, n = 8 saline, n = 9 NAFL EV, n = 7 NASH EV), NAFL (B, n = 8/group), or NASH (C, n = 8 saline, n = 9 Control EV) mice in response to saline or liver EVs across 1 cohort. P < 0.001. *, significant from Saline. (D) Blood glucose and AUC during GTT in female mice in response to saline or liver EV (n = 3/group) across 1 cohort. *, significant from NAFL EV injection. P < 0.001, P = 0.02 (inset). (E-F) Blood glucose (E, P = 0.001) and serum insulin (F, P < 0.001) during GTT in control mice that received i.v injection of saline or liver EV (n = 6 saline, n = 4 NAFL EV) across 1 cohort. *, significant from NAFL EV injection. (G-I) Blood glucose levels and the AUC (inset) of Control mice injected intraperitoneally with saline or empty liposomes (G, n = 5/group, P < 0.001 (main effect: Time), P = 0.91 (inset)), adipose-derived EVs (H, n = 7 saline, n = 6 adipose EV, P < 0.001 (main effect: Time), P = 0.76 (inset)), or serum derived EVs (I, n = 4 saline, n = 6 serum EV, P < 0.001 (main effect: Time), P = 0.603 (inset)) across 2 cohorts. (J) Serum insulin during an oral GTT in response to administration of saline (n = 6), adipose-derived EVs (n = 5), or serum EVs (n = 5) across 1 experiment. P < 0.001 (main effect: Time). (K-L) Nanoparticle-tracking analysis of liver EVs from liver slices in response to high glucose (20 mM), in the absence or presence of (K, P = 0.994) saline (n = 6), glucagon (n = 5), or norepinephrine (n = 5), and (L, P = 0.22) saline (n = 6) or insulin (n = 5) across 1 experiment for each comparison. (M) Volcano plot comparison of the liver EV proteome secreted by healthy liver slices in response to high glucose (20 mM, n = 6) relative to low glucose (2 mM, n = 5) across 1 experiment. (N-Q) Gating strategy for imaging flow cytometry in Control mice (n = 4/group) that were fasted and subjected to an oral gavage of water (vehicle) or glucose. The unfocused bright field signals were excluded using Gradient RMS feature (N). Particles with relative area smaller than the speed beads were gated by the discriminating aspect ratio and area of particles (previously optimised) (O). CD63-PE-Cy7 and CD9-APC positive particles were gated (P). ASGPR positive particles were plotted and gated based on the isotype control (Q). 1 independent experiment. Data are expressed as mean ± SEM. Each datapoint represents an independent biological sample. Data was analysed using a one-way ANOVA (A, K (calculated from AUC), repeated measures two-way ANOVA (D, E, F, G, H, I, J) with Student Newman Keuls post-hoc, or unpaired two-tailed t-test (B, C, D (inset), G (inset), H (inset), I (inset), L (calculated from AUC), M).
