Document Type : Original Article
.Cellular and Molecular Research Center, Medical Basic Sciences Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran;.Department of Anatomical Sciences, Faculty of Medicine, Ahvaz Jundishapur
.Department of Anatomical Sciences, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
.Alimentary Tract Research Center, Physiology Research Center, Medical Basic Sciences Research Institute, The School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
Destruction of pancreatic beta-cells induces an insulin deficiency and causes type 1 diabetes. The role of autophagy in inducing insulin-secreting cells (ISCs) from adipose-derived mesenchymal stem cells (AMSCs) was investigated in the current study.
Materials and Methods
In this experimental study, the isolated AMSCs were characterization and exposed to a cocktail differentiation medium (CDM) in the absence or presence of 3-methyladenine (3MA), an autophagy inhibitor. The differentiation of ISCs was confirmed by the evaluation of the expression of beta-cell-specific genes including pancreatic and duodenal homeobox 1 (PDX1), musculoaponeurotic fibrosarcoma oncogene homolog A (MAF-A), Nk class of homeodomain-encoding genes 6.1 and 2.2 (NKX6-1 and NKX2.2), Glucose transporter 2 (GLUT-2) and INSLIN. Using Newport Green (NG), insulin-positive cells were identified. Insulin secretion in response to various glucose concentrations was measured. Autophagy was evaluated by Acridine orange (AO) staining. Also, expression of autophagy-associated genes, including autophagy-related gene 5 (ATG-5), autophagy-related gene 7 (ATG-7), BECLIN-1, and mammalian target of rapamycin (mTOR), was evaluated by Real-time polymerase chain reaction (PCR) method.
We observed a significant increase of beta-cell specific genes expression in the CDM-treated cells (P < 0.01 or P < 0.001), whereas the expression of these genes was down-regulated in 3MA-exposed cells. Expression of INSULIN and GLUT-2 genes (P < 0.01 and P < 0.05, respectively), insulin secretion in response to glucose (P < 0.01), and percentage of NG-positive cells (P < 0.05) in the 3MA-exposed cells were considerably lower than the cells treated with CDM. The percentage of AO-positive cells (P < 0.01) and the expression of autophagy-related genes (P < 0.001) was significantly enhanced in the CDM group. These events were significantly prevented by the 3MA.
Our data showed that autophagy is necessary for beta-cell differentiation, and preventing autophagy by 3MA causes the reduction of beta-cell differentiation and insulin secretion.