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. 2017 Jul 3;216(7):2091–2105. doi: 10.1083/jcb.201701085

Figure 2.

Figure 2.

mTOR deficiency in β cells leads to impaired nutrient dependent insulin secretion in β cells. (A) Immunoblots showing deletion of mTOR in primary islets. Isolated islets were infected with either Ad-GFP or Ad-Cre at an MOI of 25 for 48 h. (B) Glucose-stimulated insulin secretion and ADP/ATP ratio in WT and mTOR-deficient islets (n = 4 per group). (C) Insulin secretion and ADP/ATP ratio after mTOR depletion. INS-1 cells transfected with 10 nM si-NS or si-mTOR were incubated with 3 mM glucose or 25 mM glucose in KRBH buffer for 1 h. (D) Insulin secretion in rapamycin- or PP242-treated cells. INS-1 cells were treated with either 20 nM rapamycin or 1 µM PP242 for 24 h. (E) ADP/ATP ratio in rapamycin- or PP242-treated cells. (F) Glucose-induced Ca2+ influx in WT and mTOR-deficient islets. Representative trace (left) and quantification of [Ca2+]i with Fura-2 (right) in isolated islets stimulated with the indicated amounts of glucose (n = 5 per group). (G) Mitochondrial membrane potential in WT and mTOR-deficient islets (WT, n = 5; knockout [KO], n = 4). (H) Mitochondrial membrane potential in INS-1 cells after mTOR depletion. (I) Oxygen consumption rate (OCR) was measured in mTOR-deficient islets (n = 5 per group). (J) Histogram showing ROS levels in INS-1 cells after mTOR knockdown. In C, D, E, H, and J, n = 3 independent experiments; values are given as mean ± SEM. In C, D, and E, *, P < 0.05; **, P < 0.01 versus control (si-NS or vehicle; ANOVA). In B, F, and I, *, P < 0.05; **, P < 0.01 versus WT (ANOVA). DCF, Dicholorofluorescein; TMRE, tetramethylrhodamine ethyl ester; Veh, vehicle.