Serum Extracellular Vesicles Reveal Metabolic Responses to Time-Restricted Feeding in High Fat Diet-Induced Obesity in Male Mice

Abstract

Objective: Extracellular vesicle (EV) secretion and cargo composition are dysregulated in metabolic diseases. This study aimed to identify changes in the EV size profile and protein cargoes in diet-induced obesity following time-restricted feeding (TRF) and to establish the role of EVs in obesity-related metabolic responses.

Methods: Mice were fed a high-fat diet (HFD) for 18 weeks prior to being placed either ad libitum or a time-restricted feeding for an additional 10 weeks. Mice on a normal chow ad libitum served as the control. The TRF group had food available for 10 hours and fasted for 14 hours per day.

Results: The serum EV size profile and amount displayed sex- and age-dependent changes in HFD-induced obesity, with age reducing EV amounts. HFD decreased small EV populations and increased larger EV populations, while TRF reversed these changes. Quantitative proteomic analysis showed that the abundance and composition of EV proteins changed in response to both acute stimulation with lipopolysaccharides (LPS) and HFD. Gene ontology analysis identified specific sets of EV proteins and their involved biological processes, reflecting the effect of LPS and HFD, as well as the reversal effect of TRF on metabolic and inflammatory pathways. EV proteins altered by HFD and those reversed by TRF had low protein overlap but significant functional overlap in biological processes. TRF activated the PPAR signaling pathway and the AKT-mTOR signaling pathway. The most significant impacts of HFD and TRF were observed on lipoprotein and carbohydrate metabolism, complement system, and neutrophil degranulation. The reversal effect of TRF on the complement system was pathway-specific, significantly activating the lectin complement pathway and restoring neutrophil degranulation.

Conclusion: Our data indicate that EVs are involved in diet-induced metabolic and inflammatory responses. Different EV populations may carry distinct sets of proteins involved in specific biological processes, thereby regulating diverse metabolic pathways efficiently.

Keywords: extracellular vesicles; inflammation; obesity; proteomics; time-restricted feeding.

Figure 1. Effect of HFD and TRF on serum EV profiling in middle-aged male and female mice.

A) Nanoparticle tracking analysis plots for middle-age male mice. B) Nanoparticle tracking analysis plots for middle-age female mice. C) Violin plot depicting the distribution of serum EV diameter in middle-aged female (n=3) and male mice (n=4). Two-way ANOVA revealed significance between sex and diet type. D) Violin plot illustrating the distribution of the average total number of particles of EVs per mL for middle-aged females (n=3) and males (n=4). Two-way ANOVA only showed significance between sex. Student t-test was employed for comparisons between groups. Capitalized letters indicate statistical significance between the experimental groups of male mice, whereas lowercase indicates differences within the female mice group. Different letters indicate statistical significance. *: Indicates statistical significance between the male and female mice. E) serum EV size distribution of middle-aged male mice. F) Middle-aged female experimental group EV distribution. G) Old female experimental group EV distribution. P-values for statistical analysis provided. For the EV size distribution (E-G), two-way ANOVA showed significance between sex and age. Student t-test was employed for comparisons between groups.

Figure 2. Proteomics characterization of serum EV proteins in young mice.

A) Scatter plot of the first two principal components (PC1 and PC2) derived from PCA of EV proteins in LPS (n = 3) and control (n = 3) groups. B) Volcano plot depicting EV protein characterization. C) Heatmap depicting EV protein characterization. D-K) Heatmaps depicting alterations of EV proteins in multiple pathways. For all heatmaps, the average fold change (Avg FC) of each protein was calculated as the ratio of its abundance to the average abundance across all control and LPS-treated groups.

Figure 3. Effect of HFD and TRF on serum EV protein profiles in middle-aged male mice.

A) Scatter plot of the first two principal components (PC1 and PC2) derived from the PCA of EV proteins in control (n = 3), HFD-AL (n = 4), and HFD-TRF (n = 4) groups. B) Circos plot showing overlap of altered EV proteins from three group comparisons. C) Circos plot showing shared enriched ontologies of altered EV proteins from three group comparisons. D) Enriched ontology clusters of altered EV proteins from three group comparisons. E) Protein-protein interaction network of altered EV proteins merged and pied by three group comparisons.F-H) The abundance of altered EV proteins in their respective clusters or pathways. Note: For the Circos plots (B and C), on the outside, each arc represents the identity of each EV protein list. On the inside, each arc represents an EV protein list, where each protein member of that list is assigned a spot on the arc. Dark orange color represents the proteins that are shared by multiple lists and light orange color represents proteins that are unique to that protein list. Purple lines link the same protein that are shared by multiple protein lists. The greater the number of purple links and the longer the dark orange arcs implies greater overlap among the input protein lists.

Figure 4. GSEA of altered serum EV proteins in metabolism by HFD and TRF.

A) Enrichment score plot of metabolism pathways for group comparisons of EV protein abundance between control and HFD-EVs. B) Enrichment score plot of metabolism pathways for group comparisons of EV protein abundance between control and TRF-EVs. C) Enrichment score plot of metabolism pathways for group comparisons of EV protein abundance between HFD-EVs and TRF-EVs. D-F) Corresponding heatmaps of EV protein abundance to the enrichment score plots. G) Enriched ontology clusters of altered EV proteins from three group comparisons. H-J) The abundance of altered EV proteins in their respective clusters or pathways. Note: The degree of enrichment is indicated on the figures by a normalized enrichment score (NES) (A-C). A significant positive NES value indicates that proteins in the enrichment core tend to appear towards the top of the protein set whereas a significant negative NES indicates the opposite, towards the bottom of the protein set as shown in the heatmaps (D-F).

Figure 5. GSEA of altered serum EV proteins in innate immune system by HFD and TRF.

A) Enrichment score plot of abundance of EV proteins involved in innate immune system pathway for group comparisons between control and HFD-EVs. B) Enrichment score plot of innate immune system pathway for group comparisons of EV protein abundance between control and TRF-EVs. C) Enrichment score plot of innate immune system pathway for group comparisons of EV protein abundance between HFD-EVs and TRF-EVs. D) Corresponding heatmaps of EV protein abundance to the enrichment score plots.E) Enriched ontology clusters of altered EV proteins from three group comparisons. F-I) The abundance of altered EV proteins in their respective clusters or pathways.