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To confirm the beneficial effects of 3-MA on induction of autophagy in the intact http://www.selleck.cn/products/VX-809.html animal, we used GFP-LC3 transgenic mice and examined autophagy in 3-MA-treated transgenic mice under starvation. In the control GFP-LC3 transgenic mice, few GFP-LC3 dots were observed in pancreatic acinar cells and such dots were relatively small. In the pancreatic islets, no GFP dots were detected and these islets were clearly stained for insulin (top panels, Figure 6). The GFP-LC3 structures appeared 24 h after starvation as large cup-shaped structures in both islet and acinar cells. To validate these findings, we examined both muscle (as an example of nonessential tissue) and brain (as an essential tissue) tissues by fluorescence microscopy. As shown in Figure 7(A), no GFP dots were observed in the extensor digitorum longus muscles before starvation, however, GFP-LC3 dots appeared after 24 h starvation in muscle tissues (Figure 7B). On the other hand, in brain samples, including the cerebral cortex and http://www.selleckchem.com/products/17-AAG(Geldanamycin).html medulla oblongata, no GFP-LC3 structures could be detected in spite of 24 h starvation (Figures 7D and F). In addition, islets starved for 24 h stained faintly for insulin (middle panels, Figure 6). The mean insulin staining intensity of starved islets was markedly reduced compared with that of untreated control islets, although the difference in insulin intensity was not significant (control, 61?514 �� 4364; starvation, 44?154 �� 19 925). The mean fluorescence intensity of GFP-dots was significantly higher in starved islets than untreated control islets (control, 2901 �� 576; starvation, 8306 �� 807; p http://www.selleckchem.com/products/lgk-974.html the recovered islets stained positive for insulin and the intensity of such staining was similar to the control islets, as judged by both the mean staining intensity and the merged microphotographs (lower panels, Figure 6). Finally, we assessed the effects of rapamycin on autophagy and insulin production in transgenic mice?in vivo. For this purpose, the mice were treated with 0.2 mg/kg of rapamycin intraperitoneally daily for 1, 2, 3, 4 or 5 weeks. After 1 week of such treatment, small but few dots appeared in both islets and acinar cells, however, no significant difference was observed in the pancreas of rapamycin-treated mice and rapamycin-plus-3-MA (10 mM)-treated mice (top panels, Figures 8A and B).