Glucocorticoids and checkpoint tyrosine kinase inhibitors stimulate rat pancreatic beta cell proliferation differentially.

Cell therapy for diabetes could benefit from the identification of small-molecule compounds that increase the number of functional pancreatic beta cells. Using a newly developed screening assay, we previously identified glucocorticoids as potent stimulators of human and rat beta cell proliferation. We now compare the stimulatory action of these steroid hormones to a selection of checkpoint tyrosine kinase inhibitors that were also found to activate the cell cycle-in beta cells and analyzed their respective effects on DNA-synthesis, beta cell numbers and expression of cell cycle regulators. Our data using glucocorticoids in combination with a receptor antagonist, mifepristone, show that 48h exposure is sufficient to allow beta cells to pass the cell cycle restriction point and to become committed to cell division regardless of sustained glucocorticoid-signaling. To reach the end-point of mitosis another 40h is required. Within 14 days glucocorticoids stimulate up to 75% of the cells to undergo mitosis, which indicates that these steroid hormones act as proliferation competence-inducing factors. In contrast, by correlating thymidine-analogue incorporation to changes in absolute cell numbers, we show that the checkpoint kinase inhibitors, as compared to glucocorticoids, stimulate DNA-synthesis only during a short time-window in a minority of cells, insufficient to give a measurable increase of beta cell numbers. Glucocorticoids, but not the kinase inhibitors, were also found to induce changes in the expression of checkpoint regulators. Our data, using checkpoint kinase-specific inhibitors further point to a role for Chk1 and Cdk1 in G1/S transition and progression of beta cells through the cell cycle upon stimulation with glucocorticoids.

Direct effect of glucocorticoids on glucose-activated adult rat ß-cells increases their cell number and their functional mass for transplantation.

Compounds that increase ß-cell number can serve as ß-cell replacement therapies in diabetes. In vitro studies have identified several agents that can activate DNA synthesis in primary ß-cells but only in small percentages of cells and without demonstration of increases in cell number. We used whole well multiparameter imaging to first screen a library of 1,280 compounds for their ability to recruit adult rat ß-cells into DNA synthesis and then assessed influences of stimulatory agents on the number of living cells. The four compounds with highest ß-cell recruitment were glucocorticoid (GC) receptor ligands. The GC effect occurred in glucose-activated ß-cells and was associated with increased glucose utilization and oxidation. Hydrocortisone and methylprednisolone almost doubled the number of ß-cells in 2 wk. The expanded cell population provided an increased functional ß-cell mass for transplantation in diabetic animals. These effects are age dependent; they did not occur in neonatal rat ß-cells, where GC exposure suppressed basal replication and was cytotoxic. We concluded that GCs can induce the replication of adult rat ß-cells through a direct action, with intercellular differences in responsiveness that have been related to differences in glucose activation and in age. These influences can explain variability in GC-induced activation of DNA synthesis in rat and human ß-cells. Our study also demonstrated that ß-cells can be expanded in vitro to increase the size of metabolically adequate grafts.