PMC

Primary cultures of rat islets were treated with epoxypukalide (Epox) or vehicle. Representative western-blots of cyclin D1, cyclin D2, cyclin D3, cdk4, cyclin E and cdk2 (A). Densitometric analysis of western-blots (N = 3–10) (B). (*p<0.05).

INS-1 832/13 cells were preincubated with 0.1 µM of the seven furanocembranolides (1–7) and proliferation was measured by BrdU incorporation as described in Methods section (N = 6). Proliferation was defined as the fold change above untreated cells (1.0) Threshold was over 1.5-fold increase in proliferation (A). Chemical structure of the seven furacembranolides (B).

Glucose-stimulated insulin secretion was performed in 0.1 µM epoxypukalide- or vehicle-treated rat islets as described in Methods section. 5.5 mM glucose (white bar) and 22 mmol/L glucose (black bar). Experiments were performed in triplicate (N = 9) (A). Insulin content was measured in islets treated with different concentrations of epoxypukalide (0.01–1 µM) (N = 5 in triplicate) (B) (*p<0.05).

Representative western-blot illustrating cell death protection mediated by epoxypukalide in a dose-dependent manner (A). Densitometric analysis of western-blots (N = 4) (B). Representative pictures of primary cultures of rat islets treated with epoxypukalyde (Epox) for 24 h, sections were stained for insulin (red) and TUNEL (green) and arrows indicate TUNEL-positive β-cells (C). Quantification of the percentage of TUNEL-positive β-cells (N = 6) (D). Representative western-blot illustrating the prosurvival effect of epoxypukalide (E). Densitometric analysis of western-blots (N = 5) (F). (#,&,*p<0.05).

Representative western-blot illustrating the effect of epoxypukalide on AKT proliferation pathway (A). Quantification of pAKT/AKT western-blots (N = 4) (B). Representative western-blot illustrating activation of ERK1/2 pathway (C). Quantification of pERK/ERK western-blots (N = 4) (D). Cell proliferation was induced with epoxypukalide (Epox) in rat islets pretreated in the presence of PD98059 (PD) (inhibitor or ERK1/2 pathway) or vehicle (DMSO). β-cell proliferation was measured by [³H]thymidine incorporation assay and compared to control (vehicle-treated islets) (N = 3 in triplicate) (E). (*p<0.05).

Primary cultures of rat islets were treated with 0.1 µM epoxypukalide, pukalide, leptolide, (Z)-deoxypukalide or vehicle (Ø) for 24 h. Cell proliferation was measured by [³H]thymidine incorporation as described in Methods section (N = 6 in triplicate) (*p<0.05) (A). Dose-dependence experiments to test epoxypukalide effectiveness in cell proliferation in rat islets (N = 6–12 in triplicate). Proliferation was measured by [³H]thymidine incorporation (B). Representative pictures of primary islet cell cultures treated with 0.1 µM epoxypukalide for 24 h, sections were stained for insulin (green) and BrdU (red). Arrows indicate BrdU-positive β-cells (C). Quantification of the percentage of BrdU-positive β-cells (N = 6) (*p<0.05) (D).