The Impact of Intermittent Fasting on Size Profile and Protein Cargo of Circulating Exosomes in High Fat Diet‐Induced Obese Mice

Abstract

Extracellular vesicles (EVs) have emerged as a key regulator of intercellular and inter‐organ communications, playing an important role in mediating metabolic responses. Dysregulation of exosome secretion and exosome cargo composition has been associated with metabolic diseases. However, how EVs reflect diet‐induced metabolic responses remains unexplored. The objective of the study is to identify changes in the size profile and protein cargoes of circulating exosomes in diet‐induced obesity upon intermittent fasting, as well as to establish EV biomarkers for obesity‐related dietary responses.Male mice (8‐10 weeks of age) were all fed a high‐fat diet (HFD) for 18 weeks prior to being placed either in ad libitum group (HFD‐AL) or intermittent fasting group (HFD‐IF) for an additional 10 weeks. Mice on the normal chow ad libitum (NC‐AL) served as control. The IF group had food available for 10 hours and fasted for 14 hours per day. EVs were isolated from serum and size was measured using Nanoparticle Tracking Analysis. Additionally, quantitative proteomics was conducted to identify EV protein markers and FunRich Analysis Tool was used to identify pathways related to the identified proteins.On a long‐term HFD, the IF significantly altered the size profile of serum exosomes. The HFD‐AL mice had an increasing trend in mean diameter of serum EVs by 24nm compared to the NC‐AL mice. The 10 weeks of IF caused a significant decrease in mean diameter of serum EVs by 47.9nm (P= 0.03) when compared to the HFD‐AL mice. However, neither the HFD nor IF altered the overall particle amount per unit of serum. This suggests that the population composition of EVs may be changed in response to the HFD feeding or IF. To address this question, we analyzed the specific populations of EVs with different ranges of sizes. Intriguingly, we found the HFD altered the population composition of EVs (decreased small EVs and increased large EVs) and the IF was able to reverse these changes. The mean population of EVs/exosomes (30‐150nm in size) were increased in the HFD‐IF group by ~30% (P= 0.02), whereas the populations of larger EVs (200‐350nm) were decreased by ~15% (P= 0.02) when compared to the HFD‐AL mice. Proteomic Analysis of the protein abundance within the isolated EVs showed variance between the experimental groups. Compared to the NC‐AL, the HFD‐AL group had 15 upregulated proteins (involved in pathways such as blood coagulation, immune response, and others) and 18 downregulated proteins (involved in pathways such as cytolysis, classical and alternative pathway complement activation, and others). Between the HFD‐IF and HFD‐AL groups, 43 proteins were found to be significantly different in abundance. Of those 43 proteins, 10 proteins altered by HFD‐AL were reversed by implementing IF. FunRich analysis showed that these 10 proteins belonged to the pathways involving NADP metabolism, immune responses, lipoprotein metabolism, and cholesterol homeostasis.Together, this data suggests intermittent fasting significantly alters the profile and protein cargoes of circulating EVs which may serve as an important mechanism for the metabolic benefits of intermittent fasting in obesity as well as biomarkers for obesity‐related metabolic responses.