Extracellular vesicles carrying surface-anchored adiponectin prevent obesity-related metabolic complications by enhancing insulin sensitivity

Figures

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  Figure 1. Surface-anchored Adpn-EVs (EVPP−Adpn) improve insulin sensitivity and glucose tolerance in HFD-fed mice. (A) Weight gain trajectory male (left) and female (right) HFD-fed mice after 6 weeks of i.p. injections with EVCTL and EVPP−Adpn or vehicle (PBS). (B) Insulin tolerance tests (ITT) performed in males (left) and females (right), with area over the curve (AOC) presented on the right. (C) Intraperitoneal glucose tolerance tests (GTT) in males (left) and females (right), with area under the curve (AUC) shown on the right.

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  Figure 2. EVPP−Adpn promote healthy adipose tissue remodeling. (A–C) Histological analysis of VAT in male HFD-fed mice at sacrifice. Representative hematoxylin/eosin-stained VAT sections are shown (A), from which adipocyte diameters were measured. Analysis of VAT adipocyte size distribution is presented in (B), and mean adipocyte area in (C). n = 11–12 males per group were analyzed. Scale bar, 100 μm. (D–F) Quantitive PCR gene expression analysis in VAT from male HFD-fed injected mice. mRNA expression was analyzed for the key adipogenic markers PDK4, PPARγ2, PLIN1, AdipoQ (D), for the insulin-responsive lipogenic genes ELOVL6, FAS, ACC, ACLY (E) and for the pro-inflammatory markers IL1β, MCP1, F4/80, IL6 (F). Data are presented as mean ± SEM. Dot plots represent the number of independent animals analyzed. Statistical differences were calculated using one-way ANOVA followed a Tukey's multiple comparisons test. ∗p < 0.05, ∗∗p < 0.01. Bar colors: Green, Vehicle; Red, EVCTL; Blue, EVPP−Adpn.

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  Figure 3. EVPP−Adpn alleviate liver dysfunction and improve key metabolic signaling pathways. (A–H) Histological analysis of liver sections. Representative hematoxylin–eosin (A) and picrosirius red (B) and periodic acid-Schiff (G) staining are shown for male mice after 6 weeks of treatment with EVCTL or EVPP−Adpn or vehicle (PBS). Quantification of steatosis area on HE-stained sections (C) and fibrosis area (%) on PS-stained sections (F) are presented. Total liver content of cholesterol (D), triglycerides(E),glycogen (H) have been quantified. n = 5–8 animals per group were analyzed. Scale bar, 100 μm. (I) Circulating FGF-21 in injected male mice. (J–K) Quantitative PCR analysis of hepatic gene expression in HFD-fed injected male mice. Relative mRNA levels of MASLD-associated genes (G) including steatotic (PLIN2), pro-inflammatory (IL1β, MCP1, F4/80) and fibrotic markers (TGFβ1, Col1A1). Expression of key metabolic genes (H) related to gluconeogenesis (PEPCK, G6Pase), lipogenesis (FAS, ACC) and β-oxidation (PDK4, PGC1α). (L–M) Western blot analysis of FAS (L) and ACC (Ser79) phosphorylation (M) in random-fed mice liver samples. Representative blots are shown, with GAPDH as loading control.…

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  Figure 4. EVPP-Adpnimprove tissue-specific insulin signaling via activation of the adiponectin pathway. (A–B) Western blot analysis of AKT (Ser473) (A) and AMPK (Thr172) (B) phosphorylation in liver, VAT, and skeletal muscle following insulin injection in mice. Representative blots are shown, with GAPDH as loading control. Since EVPP−Adpn and EVCTL samples were run on the same gel but not in adjacent lanes, the non-adjacent lanes were cropped for clarity. Quantification of phospho-AKT/total AKT (B) and phospho-AMPK/total AMPK ratios (B) is presented below the blots. (C) Plasma ceramide profiling showing total levels and subspecies of ceramides (left panel), dihydroceramides (middle panel) and hexosylceramides (right panel). Data are presented as mean ± SEM. Dot plots represent the number of independent animals analyzed. Bar colors: Green, Vehicle; Red, EVCTL; Blue, EVPP−Adpn.