High fructose exposure modifies the amount of adipocyte-secreted microRNAs into extracellular vesicles in supernatants and plasma

Abstract

Background: High fructose exposure induces metabolic and endocrine responses in adipose tissue. Recent evidence suggests that microRNAs in extracellular vesicles are endocrine signals secreted by adipocytes. Fructose exposure on the secretion of microRNA by tissues and cells is poorly studied. Thus, the aim of this study was to evaluate the effect of fructose exposure on the secretion of selected microRNAs in extracellular vesicles from 3T3-L1 cells and plasma from Wistar rats.

Methods: 3T3-L1 cells were exposed to 550 µM of fructose or standard media for four days, microRNAs levels were determined in extracellular vesicles of supernatants and cells by RT-qPCR. Wistar rats were exposed to either 20% fructose drink or tap water for eight weeks, microRNAs levels were determined in extracellular vesicles of plasma and adipose tissue by RT-qPCR.

Results: This study showed that fructose exposure increased the total number of extracellular vesicles released by 3T3-L1 cells (p = 0.0001). The levels of miR-143-5p were increased in extracellular vesicles of 3T3-L1 cells exposed to fructose (p = 0.0286), whereas miR-223-3p levels were reduced (p = 0.0286). Moreover, in plasma-derived extracellular vesicles, miR-143-5p was higher in fructose-fed rats (p = 0.001), whereas miR-223-3p (p = 0.022), miR-342-3p (p = 0.0011), miR-140-5p (p = 0.0129) and miR-146b-5p (p = 0.0245) were lower.

Conclusion: Fructose exposure modifies the levels of microRNAs in extracellular vesicles in vitro and in vivo. In particular, fructose exposure increases miR-143-5p, while decreases miR-223-3p and miR-342-3p.

Keywords: Adipocytes; Adipose tissue; Extracellular Vesicles; Fructose; microRNA.

Figure 1. Particle number estimation and size of EVs derived from supernatants from 3T3-L1 cells and rat plasma.

(A) The total number of particles from 3T3-L1 cells supernatants was estimated by nanoparticle tracking analysis. (B) Area under the curve from the total number of particles from 3T3-L1 cells supernatants. (C) The mean size of EVs from 3T3-L1 cells supernatants. (D) Western blot for CD63, ANXA2, and CD81 in EVs of 3T3-L1 cell supernatants. (E) Relative density of protein of CD63. (F) Relative density of protein of ANXA2. (G) Relative density of protein of CD81. (H) The total number of particles from rat plasma was estimated by nanoparticle tracking analysis. (I) Area under the curve from the total number of particles from rat plasma. (J) The mean size of EVs from rat plasma. Differences were tested by the Mann–Whitney U test. Data are presented as means ± SE.

Figure 2. Fructose exposure modified the levels of miR-143-5p and miR-223-3p in EVs, 3T3-L1 cells, and adipose tissue.

(A) Levels of miR-143-5p (black), miR-223-3p (grey) and miR-342-3p (light grey) in EVs from 3T3-L1 cells supernatant. (B) Levels of miR-143-5p (black), miR-223-3p (grey) and miR-342-3p (light grey) in EVs from rat plasma. (C) Expression of miR-143-5p (black), miR-223-3p (grey) and miR-342-3p (light grey) in 3T3-L1 cells. (D) Expression of miR-143-5p (black), miR-223-3p (grey) and miR-342-3p (light grey) in rat adipose tissue. Differences were tested by the Mann–Whitney U test. Data are presented as means ± SE.