Figure 3.

β-Cell apoptosis in primary islet cell cultures from RIP-HGF transgenic mice and normal (NL) littermates incubated at 5 mm glucose and treated for 24 h with palmitate. Representative microphotographs of (A) untreated NL, (B) 0.5 mm palmitate-treated NL, (C) 0.5 mm palmitate-treated RIP-HGF, and (D) 0.5 mm palmitate and 25 ng/ml HGF-treated NL mouse primary islet cell cultures stained for insulin (red), TUNEL (green), and Hoechst (blue). Arrows indicate TUNEL-positive β-cell nuclei. E, Quantitation of the number of TUNEL-positive β-cells in these primary islet cell cultures. F, Dose-dependent effect of HGF on the number of TUNEL-positive β-cells in primary islet cell cultures from NL mice treated with 0.5 mm palmitate for 24 h. An open circle represents untreated cells. Values are means ± sem of at least five experiments in duplicate. *, P < 0.05 vs. NL; #, P < 0.05 vs. not treated with HGF. There was no significant difference between the apoptosis levels found with 6 and 25 ng/ml HGF. G, Quantitation of the number of TUNEL-positive β-cells in primary islet cell cultures from normal mice incubated at 5 and 25 mm glucose and treated for 24 h with 0.5 mm palmitate (Palm) and/or 25 ng/ml HGF. *, P < 0.05 vs. untreated (UT); #, P < 0.05 vs. Palm+25 mm glucose; ^, P < 0.05 vs. Palm+5 mm glucose+HGF. H, Representative Western blot analyses of Akt phosphorylation (Ser473), Bad phosphorylation (Ser136) in protein extracts from RIP-HGF transgenic and NL mouse islets after treatment with or without 0.5 mm palmitate for 24 h. Samples from different parts of the same gel were used for this figure. Densitometric quantitation of several Western blottings analyzing (I) Akt phosphorylation (J) and Bad phosphorylation. The y-axis represents the ratio of phosphorylated vs. total in arbitrary units. Values are means ± sem of four different islet extracts from RIP-HGF transgenic and NL mice. *, P < 0.05 vs. NL untreated; #, P < 0.05 vs. RIP-HGF treated with palmitate.