Figure 3

Effect of glucokinase inhibition, activation or knockdown on glucagon release from single rat alpha cells measured by TIRF microscopy. (a) Cells were sequentially incubated in 4 mM glucose (4), 4 mM glucose + 10 mM d-Mannoheptulose (+ Manno) or 3 mM glucose (3) (n = 6 cells from 3 preparations; *** p < 0.001 vs 4 mM glucose, ^###p < 0.001 vs. 3 mM glucose). (b) Cells were sequentially incubated in 4 mM glucose (4), 4 mM glucose + 10 mM 2-Deoxy-d-Glucose (+ DEOXY) or 3 mM glucose (3). (n = 7 cells from 3 preparations; ***p < 0.001 vs 4 mM glucose). (c) Cells were sequentially incubated in 4 mM glucose (4), 4 mM glucose + 10 mM 5-Thio-d-glucose (+ 5-Thio) or 3 mM glucose (3) (n = 5 cells from 3 preparations; ***p < 0.001 vs 4 mM glucose). (a–c) Each incubation lasted for 4 min, with a 4 min washout between inhibitor treatment and 3 mM glucose. (d) Cells were incubated in either 4 mM glucose or 3 mM glucose sequentially without (untreated) or rising concentrations (0.1 µM, f or 10 µM) glucokinase activator RO0281675 (n = 6 or n = 11 cells from 3 preparations; ***p < 0.001 vs. untreated 4 mM glucose, ^###p < 0.001 vs. untreated 3 mM glucose). (e) Cells were transfected with negative control (control) or glucokinase siRNA (GK-KD) 5 days prior to the experiment. During imaging, cells were sequentially exposed to each of the glucose concentrations of the ramp (from 11 to 0 mM glucose) for 3 min each. (n = 12 for control or n = 9 for GK-KD cells from 3 preparations; ^###p < 0.001 vs. other glucose concentration, *p < 0.05, **p < 0.01, ***p < 0.001 for comparison between control and GK-KO). (a–e) Statistical significance was determined by one (a–c) or two (d,e) way ANOVA followed by Scheffé Means Comparison. Individual cells are depicted as symbols overlaying the boxplots with different colours indicating different preparation. The raw data can be found in Supplementary Table S1.