Figure 5. (A, B) The processes of DNA synthesis and maintenance of insulin content do not occur simultaneously.

Human islets were cultured in cCMRL, 5 or 8 mM glucose with or without the concentrations of MSDC-0160 and IGF-1 for 4 days as indicated. (A) Islet insulin content was measured from 20 islets in duplicate at the start of the incubation period (time-0) and after 4 days of incubation under the conditions shown at the bottom of the figure. Islets were sonicated in PBS +0.05% BSA and kept at −20°C overnight. Islet insulin content was measured by radioimmunoassay. Data are means±SEM from n = 3 with duplicate samples in each experiment. (B) Human islets (100) were cultured for 4 days in cCMRL, 8 mM glucose+MSDC-0160, LiCl or IGF-1 as indicated. ³H-thymidine was added to each dish 24 h before the end of the 4-day period. Data are the means±SEM from n = 3, with duplicate samples in the experiment. Data analyzed using one-way ANOVA followed by Newman-Keuls Post-hoc test. (C) MSDC-0160 decreases Wnt signaling pathway by blocking β-catenin nuclear translocation. Immunohistochemical staining of insulin and β-catenin in human islets cultured for 4 days in CMRL containing 5 or 8 mM glucose±MSDC-0160 or LiCl as indicated. Upper panel shows the merged image of β-catenin (red), insulin (green) and DAPI (blue). The white square in each image of the upper panels is enlarged and displayed in detail in the lower panel (inset). White arrows show nuclear β-catenin. Images were acquired at 40X magnification and are representative of n = 3 experiments. Scale bar = 50 µm. Insulin = green, Pdx1 = Red, DAPI = blue. (D) Sequential strategy to enhance DNA synthesis and preserve insulin content in human islets. Islets were cultured in cCMRL, 8 mM glucose+LiCl for 4-day as indicated. After a 4 day stimulatory period, medium was replaced with 5 mM glucose+MSDC-0160 and IGF-1 for another 4-day period (total 8-days of incubation). Islet insulin content was measured from 20 islets in duplicates at the start of the incubation period (time-0), after 4 days and 8 days. Islets were sonicated in PBS +0.05% BSA and kept at −20°C over night. Islet insulin content was measured by RIA. Data are means±SEM from n = 3 with duplicate samples in each experiment.