Adipose Tissue-Derived Extracellular Vesicles Loaded with miR-141-3p Regulate Obesity-Induced Insulin Resistance by Targeting Glycogen Synthesis and Gluconeogenesis

Figures

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  Figure 1. Characterization and identification of adipose tissue-derived EVs. (A) Schematic diagram of EVs isolated from adipose tissue collected from mice fed a chow or high-fat diet. Created in BioRender. Bao, X. (2025) https://BioRender.com/7ucfgo7. (B) Representative TEM images of CD-EVs and HFD-EVs. Scale bar: 200 nm. (C) The presence of marker proteins in CD-EVs and HFD-EVs. (D) PKH26-labeled CD-EVs and HFD-EVs were absorbed by AML12 cells. Scale bar: 50 μm.

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  Figure 2. Detection of miR-141-3p expression levels in different samples. (A) Detection of miR-141-3p expression in the adipose tissue of obese and lean mice by qPCR. ***P< 0.001, HFD-adipose tissue vs CD-adipose tissue. n=5. (B) Comparison of miR-141-3p levels between HFD-EVs and CD-EVs. ***P<0.001, HFD-EVs vs CD-EVs. n=5. (C) The miR-141-3p level was detected by qPCR in AML12 cells with the administration of HFD-EVs, CD-EVs, or PBS. n=3. **P<0.01 compared to the PBS administration group, #P<0.05, ##P<0.01 compared to the CD-EVs administration group. All values were expressed as mean ± SD.

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  Figure 3. miR-141-3p in adipose tissue-derived EVs regulates obesity and insulin sensitivity in mice. (A and H) Schematic diagram of mouse model construction and different EVs interventions . (A) Created in BioRender. Bao, X. (2025) https://BioRender.com/czi6cco; (H) Created in BioRender. Bao, X. (2025) https://BioRender.com/0e8x5y1. (B) miR-141-3p levels in CD-EVs-NC and CD-EVs-inhibitor were detected by qPCR. ***P<0.001, the CD-EVs-inhibitor group vs the CD-EVs-NC group. (C and D) Body weight and weekly food intake of chow diet-fed mice and high-fat diet-fed mice administrated with CD-EVs, CD-EVs-NC, CD-EVs-inhibitor, or normal saline as indicated. (E–G) The GTT, ITT, and HOMA-IR index of mice were measured after administration for 3 weeks. HOMA-IR index = [fasting blood glucose levels (mmol/L)] × [fasting serum insulin levels (μU/mL)]/22.5. **P<0.01, ***P<0.001 compared to the CD/NS group. #P<0.05, ###P<0.001 compared to the HFD/NS group. &P<0.05, the HFD/CD-EVs-inhibitor group vs the HFD/CD-EVs-NC group (I) miR-141-3p levels in HFD-EVs infected with miR-141-3p mimic or mimic NC were detected by qPCR. ***P<0.001, the HFD-EVs-mimics groups vs the HFD-EVs-NC group. (J and K) The body…

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  Figure 4. miR-141-3p in adipose tissue-derived EVs regulates insulin sensitivity in IR-AML12 cells. (A) Effects of PA on AML12 cell viability. (B) Effects on glucose uptake after treatment with PA for 24 h. *P<0.05, **P<0.01, ***P<0.001, different concentrations of PA group vs control group. (C) The levels of miR-141-3p in IR-AML12 cells after transfection of miR-141-3p inhibitor in CD-EVs. (D) Effects of glucose uptake in IR-AML12 cells after transfection of miR-141-3p inhibitor in CD-EVs. (E) The levels of miR-141-3p in different treatment groups were measured in cell after HFD-EVs were transfected with the miR-141-3p mimic. (F) Effects on glucose uptake in cell after transfection of the miR-141-3p mimic in HFD-EVs. *P<0.05, **P<0.01, ***P<0.001 compared to the Control group. #P<0.05, ##P<0.01, ###P<0.001 compared to the PA/PBS group. &P<0.05, &&&P<0.001, the PA/CD-EVs-inhibitor group vs the PA/CD-EVs-NC group or the PA/HFD-EVs-mimics group vs the PA/HFD-EVs-NC group. n=3. All values were expressed as mean ± SD.

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  Figure 5. miR-141-3p regulates PI3K/AKT signaling in hepatocytes via PTEN. (A) Effect of CD-EVs transfected with the miR-141-3p inhibitor on PTEN protein expression. (B) Effect of HFD-EVs transfected with the miR-141-3p mimic on PTEN protein expression. (C) Effect of CD-EVs transfected with the miR-141-3p inhibitor on the phosphorylation of AKT, GSK3β and FOXO1. (D) Effect of HFD-EVs transfected with the miR-141-3p mimic on the phosphorylation of AKT, GSK3β and FOXO1. *P<0.05, **P<0.01, ***P<0.001 compared to the Control group. #P<0.05, ##P<0.01, ###P<0.001 compared to the PA/PBS group. &P<0.05, &&P<0.01, the PA/CD-EVs-inhibitor group vs the PA/CD-EVs-NC group or the PA/HFD-EVs-mimics group vs the PA/HFD-EVs-NC group. n=3. All values were expressed as mean ± SD.

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  Figure 6. Adipose tissue-derived EVs regulate hepatic glycogen synthesis and gluconeogenesis through miR-141-3p. (A and B) PAS staining and liver glycogen content after transfection of CD-EVs with the miR-141-3p inhibitor. (C and D) qPCR analysis of G6PC and PCK expression in the liver after transfection of CD-EVs with the miR-141-3p inhibitor. (E) Western blotting of G6PC and PCK expression in the liver after transfection of CD-EVs with the miR-141-3p inhibitor. (F and G) PAS staining and liver glycogen content after transfection of HFD-EVs with the miR-141-3p mimic. (H and I) qPCR analysis of G6PC and PCK expression in the liver after transfection of HFD-EVs with the miR-141-3p mimic. (J) Western blotting of G6PC and PCK protein expression in the liver after transfection of HFD-EVs with the miR-141-3p mimic. *P<0.05, **P<0.01, ***P<0.001 compared to the CD/NS group. #P<0.05, ##P<0.01, ###P<0.001 compared to the HFD/NS group. &P<0.05, &&P<0.01, &&&P<0.001, the HFD/CD-EVs-inhibitor group vs the HFD/CD-EVs-NC group or the HFD/HFD-EVs-mimics group vs the HFD/HFD-EVs-NC group. n=5. Scale bar: 100 μm. All values were expressed as mean ± SD.

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  Figure 7. Effects of knocking down or overexpressing miR-141-3p in EVs on glycogen synthesis and gluconeogenesis in hepatocytes. (A and B) Effects of CD-EVs-inhibitor treatment on the PAS staining and glycogen content of hepatocytes. (C and D) Effects of CD-EVs-inhibitor treatment on G6PC and PCK mRNA expression. (E) Western blotting of G6PC and PCK protein expression after CD-EVs-inhibitor treatment of AML12 cells. (F and G) Effects of HFD-EVs-mimics treatment on the PAS staining and glycogen content of hepatocytes. (H and I) Effects of HFD-EVs-mimics treatment on G6PC and PCK mRNA expression. (J) Western blotting of G6PC and PCK protein expression after HFD-EVs-mimics treatment of AML12 cells. *P<0.05, **P<0.01, ***P<0.001 compared to the Control group. #P<0.05, ##P<0.01, ###P<0.001 compared to the PA/PBS group. &P<0.05, &&P<0.01, &&&P<0.001, the PA/CD-EVs-inhibitor group vs the PA/CD-EVs-NC group or the PA/HFD-EVs-mimics group vs the PA/HFD-EVs-NC group. n=3. Scale bar: 200 μm. All values were expressed as mean ± SD.

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  Figure 8. miR-141-3p regulates insulin signaling in the mouse liver via PTEN. (A) Effect of CD-EVs transfected with the miR-141-3p inhibitor on PTEN protein expression. (B) Effect of HFD-EVs transfected with the miR-141-3p mimic on PTEN protein expression. (C) Effect of CD-EVs transfected with miR-141-3p inhibitor on insulin signaling. (D) Effect of HFD-EVs transfected with the miR-141-3p mimic on insulin signaling. *P<0.05, **P<0.01, ***P<0.001 compared to the CD/NS group. #P<0.05, ##P<0.01, ###P<0.001 compared to the HFD/NS group. &P<0.05, &&P<0.01, the HFD/CD-EVs-inhibitor group vs the HFD/CD-EVs-NC group or the HFD/HFD-EVs-mimics group vs the HFD/HFD-EVs-NC group. n=5. All values were expressed as mean ± SD.

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  Figure 9. Schematic diagram of the mechanism revealed in this study. miR-141-3p regulates the expression of PTEN, further regulates the activation of the PI3K/AKT signaling pathway, and participates in the regulation of insulin resistance (By Figdraw).