Blocking glucose metabolism and calcium mobilization blunts glucose stimulation of JNK phosphorylation and ADAR2 expression.

<p>(<b>A</b>) High glucose did not affect the stability of <i>Adar2</i> mRNA transcripts. INS-1 cells cultured at 16.7 mM glucose for 3 hours were maintained at 2.8 or 16.7 mM glucose in the presence of 5 µg/ml actinomycin D for the indicated time intervals. The abundance of <i>Adar2</i> mRNA was analyzed by real-time RT-PCR. Results represent the mean±SEM from three independent experiments. (<b>B–C</b>) INS-1 cells maintained at 2.8 mM glucose for 12 hours were pre-cultured without serum at 2.8 mM glucose for 6 hours and then stimulated with 16.7 mM glucose in the absence or presence of 20 mM 2-deoxyglucose (2-DG, an inhibitor of glucose glycolysis) for 24 hours. (<b>B</b>) The abundance of <i>Adar2</i> mRNA was determined by real-time RT-PCR. Data are shown as the mean±SEM from three independent experiments. **<i>P</i><0.01 versus control cells at 2.8 mM glucose. (<b>C</b>) Immunoblotting was performed using the indicated antibodies. Results are representative of three independent experiments. (<b>D</b>) INS-1 cells pre-cultured at 2.8 mM glucose for 18 hours were incubated with 5 mM EGTA (a blocker of calcium influx) or 50 µM 2-APB (an inhibitor of ER calcium release through the IP3 receptor) for 30 min before stimulation by 16.7 mM glucose for 3 hours. The abundance of <i>Adar2</i> mRNA was determined by real-time RT-PCR. Data are shown as the mean±SEM from three independent experiments. *<i>P</i><0.05 versus control cells at 2.8 mM glucose. (<b>E</b>) INS-1 cells maintained at 2.8 mM glucose for 12 hours were pre-cultured without serum at 2.8 mM glucose for 6 hours and then treated for 24 hours with 100 nM insulin, or with 16.7 mM glucose in the absence or presence of 10 µM HNMPA-(AM) 3 (an inhibitor of insulin receptor tyrosine kinase activity) or 500 nM somatostatin-14 (SS-14, an inhibitor of insulin secretion). The inhibitors were added 30 min before glucose stimulation. Cell lysates were analyzed by immunoblotting with the indicated antibodies. Shown are representative results of three independent experiments.