Glucose- and Interleukin-1β-Induced β-Cell Apoptosis Requires Ca2+ Influx and Extracellular Signal-Regulated Kinase (ERK) 1/2 Activation and Is Prevented by a Sulfonylurea Receptor 1/Inwardly Rectifying K+ Channel 6.2 (SUR/Kir6.2) Selective Potassium Channel Opener in Human Islets
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Figures
- FIG. 1.
KATP channel openers protect from glucose-induced β-cell apoptosis and impaired secretory function in human islets. Human islets were cultured on extracellular matrix-coated dishes for 4 days in 5.5, 11.1, and 33.3 mmol/l glucose alone or with 3, 30, and 100 μmol/l NN414; 200 μmol/l diazoxide; or 10 and 100 μmol/l tolbutamide. A: Results are means ± SE of the percentage of TUNEL-positive β-cells per islet normalized to control incubations at 5.5 mmol/l glucose alone (100%; in absolute value, 0.32 ± 0.03 TUNEL-positive β-cells per islet). Islets were isolated from five organ donors. The mean number of islets scored from each donor was 28 (range 21–45) for each treatment condition. B: Chronic insulin release into the culture medium during the 4-day incubation period. Results are means ± SE of the insulin secreted per islet relative to control incubations at 5.5 mmol/l glucose alone (100%; in absolute value, 3.45 ± 0.59 pmol · islet−1 · day−1). C: Ratio of stimulated to basal insulin secretion during successive 1-h incubation at 3.3 (basal) and 16.7 (stimulated) mmol/l glucose after the 4-day culture period. Results are means ± SE. Islets were isolated from three organ donors. In each experiment, the data were collected from three plates per treatment. *P < 0.05 vs. control islets at 5.5 mmol/l glucose alone; **P < 0.05 vs. islets at 11.1 mmol/l glucose alone; #P < 0.05 vs. islets at 33.3 mmol/l glucose alone.
- FIG. 2.
Glucose and IL-1β activate ERK1/2 in human islets. Human islets were cultured in suspension for 30 min in 5.5 mmol/l glucose alone or in the presence of 2 ng/ml IL-1β or in 11.1 or 33.3 mmol/l glucose with or without preincubation for 1 h with 1 μmol/l PD098059, 1 μmol/l nimodipine, or 3 μmol/l NN414. Islet extracts were analyzed by Western blotting for Thr202/Tyr204-phosphorylated ERK1/2 (P-ERK1/2) and total ERK1/2 (ERK1/2) or actin. One of three experiments with all conditions, except for the presence of PD098059 and 11.1 mmol/l glucose (two experiments), from six organ donors is shown. All experiments gave similar results.
- FIG. 3.
Glucose-induced β-cell apoptosis and impaired function require l-type Ca2+ influx and ERK1/2 activation. Human islets were cultured on extracellular matrix-coated dishes for 4 days in 5.5, 11.1, and 33.3 mmol/l glucose alone or with 1 μmol/l nimodipine, 1 μmol/l nitrendipine, 1 μmol/l PD098059, or 1 μmol/l UO126. A: Results are means ± SE of the percentage of TUNEL-positive β-cells per islet normalized to control incubations at 5.5 mmol/l glucose alone (100%; in absolute value, 0.32 ± 0.03 TUNEL-positive β-cells per islet). The mean number of islets scored from each donor was 32 (range 25–43) for each treatment condition. B and C: Basal and stimulated insulin secretion during successive 1-h incubation at 3.3 (basal) and 16.7 (stimulated) mmol/l glucose after the 4-day culture period. Results are means ± SE of the insulin secreted per islet relative to control incubations at 5.5 mmol/l glucose alone (100%; in absolute value, 0.79 ± 0.08 pmol/islet for basal insulin). D: Results are means ± SE of the percentage of TUNEL-positive β-cells per islet normalized to control incubations at 5.5 mmol/l glucose alone. Islets were isolated from three (A–C) or one (D) organ donor. In each experiment, the data were collected from three plates per treatment. *P < 0.05 vs. control islets at 5.5 mmol/l glucose alone; **P < 0.05 vs. islets at 11.1 mmol/l glucose alone; #P < 0.05 vs. islets at 33.3 mmol/l glucose alone.
- FIG. 4.
IL-1β-induced β-cell apoptosis and impaired function require l-type Ca2+ influx and ERK1/2 activation and can be prevented by potassium channel openers. Human islets were cultured on extracellular matrix-coated dishes for 4 days in 5.5 mmol/l glucose or in the presence of 2 ng/ml IL-1β alone or with 1 μmol/l nimodipine, 1 μmol/l PD098059, 200 μmol/l diazoxide, or 3 μmol/l NN414. A: Results are means ± SE of the percentage of TUNEL-positive β-cells per islet normalized to control incubations at 5.5 mmol/l glucose alone (100%; in absolute value, 0.32 ± 0.03 TUNEL-positive β-cells per islet). Islets were isolated from three organ donors. The mean number of islets scored from each donor was 35 (range 31–41) for each treatment condition. B and C: Basal and stimulated insulin secretion during successive 1-h incubation at 3.3 (basal) and 16.7 (stimulated) mmol/l glucose after the 4-day culture period. Results are means ± SE of the insulin secreted per islet relative to control incubations at 5.5 mmol/l glucose alone (100%; in absolute value, 0.79 ± 0.08 pmol/islet for basal insulin). Islets were isolated from three organ donors. In each experiment, the data were collected from three plates per treatment. *P < 0.05 vs. control islets at 5.5 mmol/l glucose alone; §P < 0.05 vs. islets exposed to IL-1β alone.
- FIG. 5.
Glucose-induced IL-1β release requires l-type Ca2+ influx and ERK1/2 activation and can be prevented by a potassium channel opener. Secretion of human islets cultured in suspension for 36 h in 5.5 or 33.3 mmol/l glucose alone or in the presence of 1 μmol/l PD098059, 1 μmol/l nimodipine, or 3 μmol/l NN414. Each bar represents the means ± SE of four separate experiments with islets from four organ donors relative to control incubations at 5.5 mmol/l glucose alone (100%; in absolute values, 0.95 ± 0.22 pg · 20 islets−1 · 2 ml−1). *P < 0.05 vs. control islets at 5.5 mmol/l glucose; #P < 0.05 vs. islets at 33.3 mmol/l glucose alone.
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