Autophagy Involves in Differentiation of Insulin-Secreting Cells from Adipose Derived Stem Cells

Mahmoud Hashemitabar; Fatemeh Rezaei-Tazangi; Layasadat Khorsandi; Seyed Ali Mard

doi:10.22074/cellj.2021.7408

Autophagy Involves in Differentiation of Insulin-Secreting Cells from Adipose Derived Stem Cells

Cell J. 2021 Nov;23(6):619-625. doi: 10.22074/cellj.2021.7408. Epub 2021 Nov 23.

Authors

Mahmoud Hashemitabar^{1

2}, Fatemeh Rezaei-Tazangi², Layasadat Khorsandi^{1

3}, Seyed Ali Mard⁴

Affiliations

¹ Cellular and Molecular Research Center, Medical Basic Sciences Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
² Department of Anatomical Sciences, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
³ Department of Anatomical Sciences, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. Email: khorsandi_cmrc@yahoo.com.
⁴ Alimentary Tract Research Center, Physiology Research Center, Medical Basic Sciences Research Institute, The School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.

Abstract

Objective: 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.

Results: 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.

Conclusion: 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.

Keywords: Adipose Stem Cells; Autophagy; Differentiation; Insulin Secreting Cells.