Extracellular vesicles released by steatotic hepatocytes alter adipocyte metabolism

FIGURE 1. Primary hepatocytes from Zucker rat model. (a) Representative photographs of 10‐weeks old heterozygous Zucker lean (+/fa; ZL) and homozygous (fa/fa; Zucker fatty; ZF) rats, and of the main steps of the two step collagenase perfusion technique including manual dissociation and gauze filtration before seeding the cells into the collagenized‐plates. Note: Isolation and quality of primary hepatocytes obtained using this procedure are supported by the representative image taken under visible microscope. (b) Representative images of bodipy lipid staining of ZL‐ and ZF‐hepatocytes after 4 h in culture; size bar = 20 μm. (c) Cell viability assessed by trypan‐blue exclusion method; n = 12. The p values were denoted as follows: ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001

FIGURE 2. Ultrastructural and biochemical characterization of EVs isolated from conditioned medium of ZL and ZF hepatocytes. (a) Representative cryo‐electron micrographs of ZL‐ and ZF‐EVs; size bar = 100 nm; inserts magnified 2.5×. (b) Protein concentration of the EVs preparations measured by Bradford assay; n = 12. Each EV preparation was done from 300 millions of cells. (c) Particle number measured by NTA; n = 12. Each EV preparation was done from 300 million of cells. (d) Ratio per million of cells of protein concentration versus particle number; n = 4. (e) Size range distribution measured by NTA; n = 4. Error bars = SD. Significance was determined by t‐test between the two groups. The p values were denoted as follows: ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001

FIGURE 3. Western‐blot analysis of Zucker rat hepatocyte‐secreted EVs. Protein extracts from ZL‐ and ZF‐primary hepatocytes (10 μg) and their corresponding EVs (5 μg) were analysed by Western‐blotting using antibodies against the indicated proteins; n = 3

FIGURE 4. Density gradient analysis of EVs secreted by hepatocytes. Based on their density, EVs were separated by ultracentrifugation in a density gradient of idioxanol. Fractions were analysed by Western blotting using antibodies against the indicated proteins

FIGURE 5. Western‐blot analysis of DEPs identified by quantitative label free proteomic analysis. Three independent preparations of EVs from Lean and Obese Zucker rat hepatocytes (10 μg) were analysed by Western blotting by using antibodies against indicated proteins

FIGURE 6. Cellular uptake and energetic effect of hepatic‐EVs on 3T3‐L1 adipocytes. (a) Confocal microscopy uptake analysis of EV‐treated 3T3‐L1 adipocytes. Cells were incubated in the presence of 10 μg of Dil‐labelled ZL‐EVs and ZF‐EVs. CTRL corresponds to 3T3‐L1 adipocytes incubated with DiI containing media after ultracentrifugation. Nucleus are stained with DAPI. (b) Individual parameters of glycolysis stress test on EV‐treated (10 μg) 3T3‐L1 adipocytes, n = 3; One‐way ANOVA (F(2, 37) = 2.426, p = 0.1023)

FIGURE 7. Effect of hepatic‐EVs on 3T3‐L1 adipocytes glucose uptake and lipolysis. (a) Quantification of the fold increase in 2‐deoxy‐glucose uptake in 3T3‐L1 adipocytes that were incubated in absence or presence 15 μg of EVs from ZL‐ and ZF‐EVs during 24 h. (b) Glycerol release following 2 h stimulation with 10 μM norepinephrine of 3T3‐L1 adipocytes untreated (PBS) or treated for 16 h with 5 μg EVs from ZL or ZF hepatocytes. Data is shown relative to untreated, unstimulated cells. n = 2; error bars = SD calculated as s = |x2−x1|/√2. The p values were denoted as follows: ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001

FIGURE 8. Metabolomics analysis of 3T3‐L1 adipocytes treated with ZL‐ and ZF‐EVs. (a) Scores OPLS‐DA plots for short (1 × 10 μg for 24 h) and long (3 × 10 μg every 24 h) treatments. Samples have been coloured depending on their class (CTRL, ZL, and ZF) in green, orange, and red, respectively. (b) Boxplots summarizing the differences per sample groups and treatment for the 12 identified metabolites in short treatment. (c) Boxplots summarizing the differences per sample groups and treatment for the 12 identified metabolites in long treatment. Adjusted p‐values for both ANOVA and pairwise t‐tests are indicated in the corresponding group comparisons