Thursday September 23, 2021 - 15:00 to 16:15
Uncovering the porcine insulin secretion pathway
Kieran Purich1,2,3,4, Jim Wickware1,2,3,4, Gina Rayat1,2,3,4.
1Department of Surgery, University of Alberta, Edmonton, AB, Canada; 2Alberta Diabetes Institute, University of Alberta, Edmonton, AB, Canada; 3Alberta Transplant Institute, University of Alberta, Edmonton, AB, Canada; 4Ray Rajotte Surgical-Medical Research Institute, University of Alberta, Edmonton, AB, Canada
Introduction: Islet transplantation is a potential alternative treatment for type 1 diabetics; however, its widespread use is limited due to the scarcity of human pancreas donors. One potential solution to such is porcine islet xenotransplantation. Success with reversal of diabetes has been demonstrated with porcine islet transplants in non-human primates; however, many barriers still prevent its clinical application. Limited study has been performed on porcine islet cell signaling pathways, which may differ from the mechanisms in humans and rodents. Studying porcine islet gene expression in the early post-natal phase could help us better understand pancreatic endocrine ontogenesis. In order to investigate such, we identified molecules involved in the insulin secretion pathway including the glucose transporter GLUT2, GTPase RAC1 which is involved in F-actin remodeling in islet cells, and SNAP-25, a protein involved in insulin release. We evaluated the relationships of these molecules with common cadherins associated with cell-cell adhesion and with islet hormone secretion.
Method: Islets from pigs 3(n=4) and 10(n=3) days after birth were isolated and samples were collected on days 1,3,5 and 7 of culture to quantify trends in GLUT2, SNAP-25, RAC1, E-, N- and VE-cadherin gene expression by qRT-PCR. Immunofluorescence staining was performed to localize protein expression.
Results: Qualitatively, the islets from both ages appeared similar on days 1,3,5 and 7 of culture. Similar trends in RAC1, SNAP 25, E-, and VE-cadherin gene expression were observed in islets from 3 and 10-day-old pigs. RAC1 gene expression was relatively constant across the 7 days of culture while maximum SNAP 25 and E-cadherin gene expression occurred at day 5 of culture in islets from both ages. VE-cadherin expression in islets from both groups followed a downward trend with maximum gene expression on day 1 of culture. Different trends in GLUT2 and N-cadherin expression were observed between groups. Maximum GLUT2 gene expression occurred at day 3 and day 7 of culture for islets from 3 and 10-day-old pigs, respectively. The gene expression of N-cadherin reached maximum at day 7 and day 5 of culture for islets from 3 and 10-day-old pigs, respectively. In general, the changes in gene expression levels of these molecules were lower in islets from 10-day-old pigs compared to islets from 3-day-old pigs. Preliminary immunofluorescence staining showed co-localization of SNAP25 and insulin proteins in islets from both ages of pigs.
Discussion/Conclusion: Our data showed similarities in RAC1, SNAP25, E- and VE-cadherin gene expression in islets from 3 and 10-day old pigs while the expression patterns of GLUT2, and N-cadherin differ between these two groups. These findings may be important for the development of porcine islets in vitro and warrant further investigation of these molecules at various post-transplant periods.
Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant. Canadian Institutes of Health Research (CIHR) - Canadian Graduate Scholarships - Master's Program. Alberta Graduate Excellence Scholarship (AGES). Walter H. John's Graduate Fellowship (University of Alberta). Royal College of Physicians and Surgeons of Canada (RCPSC) - Clinician Investigator Program.