Pancreatic stellate cells promote pancreatic β-cell death through exosomal microRNA transfer in hypoxia

Hypoxia in pancreatic islets (islet hypoxia) can occur in type 2 diabetes mellitus. Previously, our in vitro experiments demonstrated that pancreatic stellate cells (PSCs) within the islet are activated in hypoxia, promoting pancreatic β-cell death. Here, we aimed to demonstrate the in vivo activation of intra-islet PSCs and investigate the mechanism of PSC-induced β-cell death in hypoxia. A novel in vivo model of islet hypoxia was established by injecting fluorescent microspheres into a carotid artery of Balb/c mice (Microsphere mice). The intraperitoneal glucose tolerance (IPGTT) was performed, and pancreatic tissues were stained for insulin expression after tissue clearing. Pimonidazole staining was also performed in the pancreas to detect the presence of hypoxia in islets. Next, primary PSCs were isolated and cultured from Balb/c mice. Exosomes were isolated from culture media from PSCs cultured in hypoxia (1% oxygen). MicroRNAs (miRNAs) were prepared from exosomes from PSCs, and miRNA expression profiles were analyzed by miRNA sequencing. Several miRNAs were overexpressed in islets using miRNA mimics. Two weeks after injection of microspheres, the Microsphere mice showed worsening of glucose tolerance in IPGTT. Later, cataracts were developed in the eyes of the mice. The pancreas showed that the areas, perimeters, and diameters of insulin-positive cells decreased in Microsphere mice. Pimonidazole adducts were detected in the islets of these mice, indicating the presence of islet hypoxia. In addition, α-smooth muscle actin-positive cell numbers per islet were higher in Microsphere mice, confirming the in vivo activation of intra-islet PSCs in hypoxia. Mouse islets incubated with exosomes isolated from PSCs cultured in hypoxia showed a decrease in cell viability. The exosomes contained a variety of miRNAs, of which miR-23a-3p was found to notably increase β-cell death through apoptosis. Together, our in vivo and in vitro data provide evidence to support that PSCs within the islets are activated in hypoxia and promote β-cell death through exosomal miRNA transfer, which may contribute to the progression of type 2 diabetes mellitus.