Adipose stem cells-derived small extracellular vesicles transport Thrombospondin 1 cargo to promote insulin resistance in gestational diabetes mellitus

Abstract

Background: Gestational diabetes mellitus (GDM) is a highly prevalent disease and poses a significant risk to the health of pregnant women. Abdominal adipose tissue (AT) contributes to insulin resistance (IR) associated with GDM. However, the underlying mechanisms remain unclear.

Methods: In this study, we developed a mouse model of GDM by subjecting mice to a high-fat diet. We collected adipose-derived stem cells (ADSCs) from the abdominal and inguinal regions and examined their role in inducing IR in normal tissues through the secretion of small extracellular vesicles (sEVs). The sEVs derived from ADSCs isolated from GDM mice (ADSC/GDM) were found to inhibit cell viability and insulin sensitivity in AML12, a normal mouse liver cell line.

Results: Through proteomic analysis, we identified high levels of the thrombospondin 1 (Thbs1) protein in the sEVs derived from ADSC/GDM. Subsequent overexpression of Thbs1 protein in AML12 cells demonstrated similar IR as observed with ADSC/GDM-derived sEVs. Mechanistically, the Thbs1 protein within the sEVs interacted with CD36 and transforming growth factor (Tgf) β receptors in AML12 cells, leading to the activation of Tgfβ/Smad2 signaling. Furthermore, the administration of LSKL, an antagonistic peptide targeting Thbs1, suppressed Thbs1 expression in ADSC/GDM-derived sEVs, thereby restoring insulin sensitivity in AML12 cells and GDM mice in vivo.

Conclusions: These findings shed light on the intercellular transmission mechanism through which ADSCs influence hepatic insulin sensitivity and underscore the therapeutic potential of targeting the Thbs1 protein within sEVs.

Keywords: Adipose-derived stem cells; Gestational diabetes mellitus; Insulin resistance; Small extracellular vesicles; Thrombospondin 1.

Fig. 1

Isolation, culture and purification of GDM mice-derived ADSCs and their sEVs. A Plasma glucose values were quantified in eight normal gestation and GDM mice. B The expression of insulin, leptin, adiponectin and hs-CRP in the plasma of mice was detected by ELISA assays. C ADSCs was obtained by collagenase digestion of abdominal and inguinal adipose tissue from gestational mice and further in vitro culture. D Microscopic images of ADSCs isolated and cultured from normal gestational and GDM mice. Scale bar, 25 μm. E The expression of Cd44 marker (red) was identified by immunofluorescence. DAPI staining (blue) indicated the nucleus. Scale bar, 25 μm. F The sEV^A and sEV^AG were identified by transmission electron microscope. Scale bar, 100 nm. G The sEV^A and sEV^AG solution was diluted 500 times and particle size distribution and concentration were determined by nanoparticle tracking analysis. H The sEV markers and stem cell markers were detected by western blotting in sEV^A and sEV.^AG samples. Data were presented with mean ± standard deviation (SD), *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 2

ADSC/GDM-derived inhibits hepatocyte proliferation and insulin sensitivity in normal mice. A, B The sEV^A and sEV^AG with different concentrations were co-cultured with AML12 cells for 48 or 72 h. Cell viability was obtained by CCK8 assays. C, D The sEV^A and sEV^AG with 10⁸ particles / mL were co-cultured with AML12 cells for 48 or 72 h. Cell apoptosis was detected by flow cytometry. E, F The sEV^A and sEV^AG with 10⁸ particle number / mL were co-cultured with AML12 cells for 48 or 72 h, then cells were treated with 1 μg/ml insulin for 1 h. Glucose uptake was measured to reflect insulin sensitivity of AML12 cells in each treatment. G The sEV^A and sEV^AG with 10.⁸ particle number /mL were co-cultured with AML12 cells for 48 h, and mRNA expression of endoplasmic reticulum (ER)-related genes was detected by RT-PCR. H The expression of ER-related proteins was detected by western blotting. Data were presented with mean ± standard deviation (SD); ns, no significance; *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

Proteomic identification of high Thbs1 expression in ADSC/GDM-derived sEV. A Venn diagram of the number of proteins detected by unlabeled protein profiles for and sEV^A (AD in graph) and sEV^AG (ADGDM in graph). B The number of proteins with high and low expression in sEV^AG. C Volcanic map of differentially expressed protein in sEV^AG. D GO signaling pathway analysis of sEV^A and sEV^AG proteins. E KEGG signal analysis of sEVA and sEV^AG proteins. F GSEA analysis revealed enrichment of the ECm-receptor interaction pathway. G Network analysis of interactions between differential ECM regulatory proteins and other differential proteins. H. Heat maps of 10 proteins significantly overexpressed in sEV^AG. I, J Mass spectrometry quantification and western blotting of Thbs1 in sEV^A and sEV.^AG. ***P < 0.001

Fig. 4

Overexpression of Thbs1 inhibits insulin sensitivity in AML12 cells. A Flag-eGFP-Thbs1 plasmid and control vector were transfected into AML12 cells. The expression of eGFP-thbs1 fusion protein in AML12 was identified by western blotting and fluorescence microscopy. B Plasmid transfected AML12 cells were placed in 96-well plates, and cell viability was detected by CCK8 assay. C. Apoptosis assays of AML12 cells after plasmid transfection were performed. D Plasmid transfected AML12 cells were treated with 1 μg/ml insulin for 1 h, and the culture medium was collected for glucose uptake measurement. E–G AML12 cells were treated with 10⁸/mL sEV^AG, 10 μM LSKL and 10⁸/mL sEV^AG + 10 μM LSKL for 48 h. Cell viability, apoptosis, and insulin-induced glucose uptake were then measured. H The expression of endoplasmic reticulum stress (ERS)-related mRNA in AML12 cells after plasmid transfection was detected by RT-PCR. I The expression of ERS-related mRNA in 10⁸/mL sEV^AG and/or 10 μM LSKL treated AML12 cells was detected by RT-PCR. J The expression of ERS-related proteins in AML12 cells was detected by western blotting. Statistical data were presented as mean ± standard deviation (SD); ns, no significance; *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

ADSC/GDM-derived sEV carrying Thbs1 induces Tgfβ and CD36 activation. A sEV^AG was co-cultured with AML12 for 48 h, and the expression of Smad2, phosphorylated Smad2 and Tgfβ was detected by western blotting. B sEV^AG was co-cultured with AML12 for 48 h, and the cell lysate was subsequently subjected to co-immunoprecipitation (co-IP) with anti-Tgfβ antibodies. Thbs1 levels in co-IP products and input samples were detected. C, D AML12 cells were treated with 108 /mL sEV^AG, 2 μM ITD-1, 10 μM LSKL or a combination for 48 h. The expression of Smad2 and phosphorylated Smad2 was detected by western blotting. E Cell lysate was co-immunoprecipitated with anti-CD36 antibody. Thbs1 levels in co-IP products and input samples were detected. F AML12 cells were treated with 10⁸/mL sEV^AG, 10 μM LSKL or a combination for 48 h, followed by co-IP with anti-CD36 antibody

Fig. 6

ADSC/GDM sEV restore insulin sensitivity to liver cells after Thbs1 pharmacological interference. A ADSC-GDM cells were treated with different concentrations of LSKL (0, 5 μM, 10 μM) for 72 h. sEV^AG, sEV^AG−L5 and sEV^AG−L10 were collected and identified by TEM and NTA. B The expression of Thbs1 in sEV^AG, sEV^AG−L5 and sEV^AG−L10 was identified by western blotting. C sEV^AG, sEV^AG−L5 and sEV^AG−L10 were co-cultured with AML12 for 24–96 h, and cell viability was detected by CCK8 assays. D sEV^AG, sEV^AG−L5 and sEV^AG−L10 were co-cultured with AML12 for 48 h, and cell apoptosis was detected by flow cytometry. E Glucose uptake of AML12 cells after insulin treatment was measured. F. sEV^AG, sEV^AG−L5 and sEV^AG−L10 were co-cultured with AML12 for 48 h, and the expression of phosphorylated Smad2 and total Smad2 protein was detected by western blotting. G, H RT-PCR and western blotting were used to detect the expression of ER stress related genes in AML12 cells. Statistical data were presented as mean ± standard deviation (SD); ns, no significance; *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 7

Targeted Thbs1 pharmacological therapy improves insulin resistance phenotype in GDM mice. A Plasma was extracted from normal, GDM, and LSKL-treated GDM mice, and blood glucose and insulin, leptin, adiponectin and hs-CRP levels were measured by glucose meter and ELISA kit. B HE staining and immunohistochemical detection of adipose tissues from normal, GDM, and LSKL treated mice. C HE staining and immunohistochemical detection of liver tissues from these mice. Statistical data were presented as mean ± standard deviation (SD); ns, no significance; *P < 0.05, **P < 0.01, ***P < 0.001