Brown adipocyte-derived exosomes in type 2 diabetes mellitus impair endothelial function via regulating intracellular calcium cycle

Figures

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  Figure 1. Extraction and identification of exosomes derived from adipose cells. (A) Epididymal fat, perirenal fat, subcutaneous fat and brown fat were clipped from diabetic mice and non-diabetic mice, and the culture medium supernatant was obtained for exosomes extraction. (B) The particle size of the lipogenic exosomes extracted in this experiment was detected by NTA experiment, and the particle size was judged to be between 115 nm and 165 nm, n = 3. (C) Particulate matter in the form of exosomes vesicles was detected from the suspension containing lipogenic exosomes by TEM observation, n = 3. (D) exosomes’ marker proteins were detected by western-blot. CD81, CD63 and HSP70 were detected as positive marker proteins of exosomes, n = 3.

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  Figure 2. Co-culture of adipocyte derived exosomes and mice vascular aorta. (A–D) The vasorelaxation function of ACh treated mice co-cultured with different type of AT-exosomes from diabetic mice and non-diabetic mice. The differences were statistically significant between experiment groups, *p ≤ 0.05, **p ≤ 0.01, n = 3. (E–H) Effect of AT-exosomes from diabetic mice on vasorelaxation after the addition of SNP, compared with non-diabetic mice. (I) The difference of vasorelaxation in vascular aorta cocultured with AT-exosomes from different adipose tissue and PBS. ***p ≤ 0.001, n = 3. (G) After the addition of SNP, the vasorelaxation of vascular aorta co-cultured with AT-exosomes from different adipose tissue and PBS, n ≥ 3.

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  Figure 3. Cytological verification of vascular endothelial dysfunction caused by AT-exosomes. (A–D) The phosphorylation levels of eNOS in vascular endothelial cells were detected after the collection of AT-exosomes (db/db and db/m+) and co-culture with MAECs and HAECs. The changes in eNOS phosphorylation levels were different, *p < 0.05 and * *p < 0.01, n = 3. (E–H) The changes of eNOS activity and NO secretion in MAECs and HAECs were tested. The differences between the experimental group and the control group (PBS) were showed by *p < 0.05 and * *p < 0.01, n = 3.

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  Figure 4. Gene sequencing of MAECs co-cultured with BAT-derived exosomes from diabetic mice and non-diabetic mice. (A–D) AT-exosomes from diabetic mice and non-diabetic mice served as controls to compare differential genes in MAECs. (E) Genes associated with cell proliferation were selected and qPCR was used to verify whether there were differences in the expression levels of target genes. *p < 0.05, ***p < 0.001, n ≥ 3.

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  Figure 5. The expression levels of CAMKII phosphorylation in MAECs and HAECs were detected. (A–D) CAMKII phosphorylation level expressed different in MAECs and HAECs co-cultured with AT-exosomes, * *p < 0.01, n = 3.

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  Figure 6. ITPR3 regulates Ca2+/CAMKII/eNOS/NO signaling pathways. (A) ITPR3 was verified whether down-regulated or up-regulated in MAECs, n ≥ 3. (B–D) The concentration of calcium ions in cytoplasm and mitochondria was stained with fluorescent dye, n ≥ 3. (E–H) The phosphorylation levels of eNOS and CAMKII in the cell model were detected. (I, J) The changes of NO secretion content and eNOS activity in the cell model were detected, *p < 0.05, **p < 0.01, ** *p < 0.001 indicates statistical significance, n ≥ 3.