Congenital Hyperinsulinism; Effects of Rapamycin on Min6 Pancreatic β-Cell Line

Abstract

Introduction: Congenital Hyperinsulinism (CHI) is a rare neonatal syndrome associated with continuous inappropriate insulin secretion by the pancreatic β-cell in the presence of recurring hypoglycemia. Newborn babies with CHI present with hypoglycemia and often do not respond to medical therapy. Although rapamycin has been used to successfully treat a small number of CHI patients, the mechanism of action to reduce insulin secretion is still unclear. The aim of this study was to assess the effects of rapamycin on cell proliferation, apoptosis, insulin secretion and changes in intracellular Ca2+ concentration ([Ca2+]i)in Min6 mouse insulinoma β-cell line, an in vitro model system that reflects many properties of normal human β-cells. Methods: Cells were first checked for genetic expression of signaling components required for rapamycin action using RT-PCR. Cell proliferation rate and viability were evaluated by treating the cells with ascending concentrations of rapamycin (0- 300 nM) followed by manual cells count using trypan blue dye exclusion during the 4 days of treatment. Cells were checked for apoptotic events via measuring Caspase 3/7 activity following 3 days of treatment with 200 nM rapamycin. Insulin secretion was assessed using ELISA following stimulation with glucose (20 mM), ATP (100 μM), UTP (100 μM), ACh (100 μM) and diazoxide (200 μM) in the presence or absence of short- or longer-term exposure to rapamycin (200 nM). Finally, the effect of rapamycin on [Ca2+]i was assessed using FlexStation3 after cells were stimulated with ATP+UTP (100 μM each) , ACh (100 μM) and KCl (40 mM) (with and without rapamycin) .Results: Rapamycin reduced cell proliferation at every concentration used with significant reduction of cell numbers following 4 days of treatment. Treating the cells with rapamycin (200 nM) for 3 days had no detectable caspase activity in Min6 cells. High glucose concentration, ATP, UTP and ACh all elicited robust increases in insulin secretion from Min6 cells. Rapamycin significantly inhibited glucose- and ATP/UTP/ACh-stimulated insulin secretion back to basal levels. Pre-incubation of Min6 cells in rapamycin prior to insulin secretion experiments resulted in reduced insulin secretion. However, rapamycin did not prevent increases in [Ca2+ ]i levels following stimulation with ATP+UTP and ACh.Conclusion: Rapamycin did not influence changes in intracellular [Ca2+]i brought about by different stimulators of insulin release. This could mean that rapamycin might be involved in the inhibition of later events in insulin signaling such as recruitment of insulin granules from the reserve pool (Ca2+-dependent), granule movement or docking with the cell membrane. Therefore, further investigations are required to elucidate the mechanism of how rapamycin inhibit insulin secretion.

Date of Award	1 Aug 2017
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Xin Wang (Supervisor) & Karen Cosgrove (Supervisor)