Fig. 6.

The cytoprotecitve effect of genistein on β-cells was dependent of GPR30-mediated activation of CREB. Western blot analyses of phosphorylated (p-CREB) and total CREB in INS1 cells (A) or human islets (B) incubated with various concentrations of genistein or vehicle for 30 min. (C) Total CREB and p-CREB protein levels in INS1 cells pretreated with PKA inhibitor H89 (10 μM) or vehicle (V) for 30 min, followed by addition of genistein (G, 5 μM) for 30 min. (D) INS1 cells transfected with reporter plasmid pCRE-Luc were incubated with vehicle or genistein for 24 h. Luciferase activity was measured and normalized to the control plasmids in the cell extracts. (E) INS1 cells were transfected with vehicle (V), scrambled siRNA (S), or siRNA directed against CREB (I) for 48 h. The efficiency of knockdown in transfected cells was verified by immunoblot analysis of CREB protein levels. (F) Total and p-CREB levels in control or transfected INS1 cells treated with genistein (G, 5 μM) or vehicle (V) for 30 min. (G) Bcl-2 protein levels in control or transfected INS1cells treated with genistein (G, 5 μM) or vehicle (V) for 48 h. (H) Quantification of apoptosis in control or transfected INS1 cells following treatment with 0.5 mM palmitate (PA) and high glucose (Glu, 20 mM) or vehicle in the presence or absence of 5 μM genistein (G) for 72 h. I. INS1 cells were transfected with scrambled control siRNA (S) or GPR30 siRNA (I). 48 h later, genistein (5 μM) effect on p-CREB and total CREB protein contents in cells were measured by western blot. (J) total CREB and p-CREB levels in WT and GPR30^−/− (KO) islets isolated from male mice and treated with or without genistein (G, 5 μM) for 30 min. (K) Cellular caspase-3 activity in WT and KO islets incubated with palmitate (PA, 0.5 mM) plus 20 mM glucose (Glu) in the presence or absence of genistein (G, 5 μM) for 24 h. Values are mean ± SEM (n=4). *, p< 0.05.