Résumé
Background and aims: Type 2 diabetes (T2D) is characterized by a chronic hyperglycaemia resulting from both a defect in pancreatic β-cell function and an insulin resistance of peripheral tissues (skeletal muscles and adipose tissue) and the liver. T2D is often associated with overweight and obesity, and the increased visceral adipose tissue is a major source of pro-inflammatory cytokines involved in the pathogenesis of the disease. In the present study, we investigated whether human visceral adipose tissue can secrete extracellular vesicles (EVs) with differentially expressed miRNAs according to the metabolic status of the patients and thereby may affect the function of other tissues involved in glucose homeostasis.Materials and methods: Fifteen morbidly obese patients were included in the clinical research protocol “COMET” conducted by our University Hospital. Five of them were insulin sensitive (IS; HOMA IR<3), insulin resistant (IR; HOMA IR≥3), or recently diagnosed type 2 diabetic (T2D) patients and were matched with age and sex. Visceral adipose tissue was obtained during bariatric surgery and used to produce conditioned media after 24 hours culture. Extracellular vesicles (EVs) were isolated by precipitation using the Total Exosome Isolation kit. The size of the vesicles was analyzed by Dynamic Light Scattering (DLS) on a Zetasizer Nano ZS. RNA content of EVs was purified and used to construct a small RNA bank using the NEXTflex Small RNA-Seq kit. miRNAs content were identified and quantified by small RNA sequencing (HiSeq 2500, Illumina).Results: Isolated EVs analyzed by DLS revealed an average size of 80 nm, with a population of higher size, probably reflecting the presence of vesicle aggregates. Analysis of markers of EVs by Western blotting revealed the presence of tetraspanins CD63, CD9 and CD81, and also the soluble proteins Hsp-70 and β-actin. After extraction of RNAs from EVs, we identify the presence of a population of miRNAs with an average length of 20nt on Bioanalyzer small RNA chip. The presence of miRNAs was confirmed with real-time PCR by the selective amplification of miR15a, miR30a, and RNU6B. Small RNA sequencing revealed the presence of 9 miRNAs differentially expressed between IS and DT2 patients, 18, between IR and DT2 patients, and 11, between IS and IR patients. Among miRNAs identified, miR-155-5p and miR-150-5p were found overexpressed in T2D patients as compared to IS (respectively by 11.83 and 4.29-fold) or IR subjects (respectively by 7.58 and 7.25-fold) (P< 0.05). Another miRNA-X, having no homolog in rodents, was diminished by 114-fold in diabetic patients as compared to IS and IR subjects (P<0.05). An additional miRNA-Y, having a homolog in rats, was at the opposite, overexpressed by 4.73 and 3.9-fold in diabetic patients as compared to IS and IR respectively (P<0.05).Conclusion: In the present study, we have demonstrated that visceral adipose from obese patients displaying different metabolic status, can secrete small EVs. Pattern of differentially expressed miRNAs has been identified, that could be involved in the pathogenesis of T2D.