Date Published: May 29, 2019
Publisher: Public Library of Science
Author(s): N. A. Garcia, H. González-King, E. Grueso, R. Sánchez, A. Martinez-Romero, B. Jávega, J. E. O’Connor, P. J. Simons, A. Handberg, P. Sepúlveda, Gangjian Qin.
Regulation of circulating free fatty acid (FFA) levels and delivery is crucial to maintain tissue homeostasis. Exosomes are nanomembranous vesicles that are released from diverse cell types and mediate intercellular communication by delivering bioactive molecules. Here, we sought to investigate the uptake of FFAs by circulating exosomes, the delivery of FFA-loaded exosomes to cardiac cells and the possible role of the FFA transporter CD36 in these processes. Circulating exosomes were purified from the serum of healthy donors after an overnight fast (F) or 20 minutes after a high caloric breakfast (postprandial, PP). Western blotting, Immunogold Electron Microscopy and FACS analysis of circulating exosomes showed that CD36 was expressed under both states, but was higher in postprandial-derived exosomes. Flow cytometry analysis showed that circulating exosomes were able to take-up FFA directly from serum. Importantly, preincubation of exosomes with a blocking CD36 antibody significantly impeded uptake of the FFA analogue BODIPY, pointing to the role of CD36 in FFA exosomal uptake. Finally, we found that circulating exosomes could delivery FFA analogue BODIPY into cardiac cells ex vivo and in vivo in a mice model. Overall, our results suggest a novel mechanism in which circulating exosomes can delivery FFAs from the bloodstream to cardiac tissue. Further studies will be necessary to understand this mechanism and, in particular, its potential involvement in metabolic pathologies such as obesity, diabetes and atherosclerosis.
Circulating free fatty acids (FFAs) are thought to be the major source of lipid fuel in the body, and are crucial to the energy metabolism of the renal cortex , myocardium , liver , and resting skeletal muscle . It is well recognized that circulating FFAs are almost exclusively derived from the adipose tissue (AT) through the hydrolysis of triglycerides (TGs) . Endothelial lipases, principally lipoprotein lipase (LPL), also contribute to the circulating FFA pool, especially after ingestion of a fat meal, by hydrolysing circulating TGs carried in chylomicrons in the capillaries of AT. For the most part, released FFAs are taken-up by adipocytes for storage, but a proportion generally escapes and contributes to the circulating FFA pool  in a process called spillover. This process may constitute 40–50% of the total circulating FFAs in the postprandial period .
The ethics committee of the IISLaFe, Valencia, Spain, reviewed and approved the study that we are presenting. Approval number: 2016/0763. Form of obtained: oral.
Circulating exosomes isolated from fasting (F) and postprandial (PP) states were visualized by electron microscopy, revealing a typical bi-layered spheroidal shape and size (Fig 1A). Also western blotting of extracts detected equal levels of the common tetraspanin markers CD9, CD81 and CD63 (Fig 1B). Anti-CD63 Immunogold electron microscopy images are shown in S1 Fig. Counting and sizing of exosomes on the NanoSight instrument revealed a different size distribution between F and PP states (Fig 1C and 1D), with larger exosomes under the F state. No differences were found in the quantity of exosomes between the two states (Fig 1E).
Circulating FFAs are crucial as energy substrates and for the synthesis of most lipids. Nevertheless, oversupply of FFAs disrupts cellular acid-base homeostasis, alters the integrity of cellular membranes and elicits the generation of harmful bioactive lipids . Thus, the regulation of circulating FFA is fundamental to the physiology of tissues. In the fasting state, the principal source of FFAs to most tissues originates from AT lipolysis. After a meal, fats are carried by chylomicrons to the bloodstream for final storage in the AT and this process generates FFA spillover, which contributes to the circulating FFA pool . Our results allow us to propose a role for circulating exosomes in FFA delivery from the bloodstream to tissues.