VAMP4 deficiency causes increases in intracellular (pro)insulin and insulin release. (A–D) Maximum intensity projection images showing the intensity of VAMP4 (green), proinsulin (red; A), and insulin (red; C) in β cells from WT and KO mice. Quantification of the proinsulin intensity (B) and insulin intensity (D) in whole cells based on the images shown in A and C. The data in B and D are shown as the mean ± SEM (n = 50 and 70 cells from four biological independent experiments, n = 4, 1 female and 3 male mice at 16 wk of age). Scale bars, 2 μm. (E–H) Representative confocal images costained for proinsulin (gray), insulin (red), and VAMP4 (green; E) in WT and KO INS-1 cells. The proinsulin intensity (F), insulin intensity (G), and ratio of proinsulin to total insulin (proinsulin and insulin; H) were quantified for maximum intensity projection based on the immunofluorescence staining data shown in E. Four sections of WT cells and five sections of KO cells were analyzed from three biological independent samples. Scale bars, 5 μm. (I) Measurement of insulin secretion from islets isolated from WT and KO mice incubated for 1 h in 2.8 or 16.7 mM glucose. 50 islets were used in each experiment, and three biological experiments were performed (n = 3, 1 female and 2 male mice at 16 wk of age). (J) Measurement of insulin secretion from WT, KO, and rescued INS-1 cells incubated for 1 h in 2.8 or 16.7 mM glucose (n = 6 biological independent samples). The data are presented as the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001. The statistical analyses were performed with two-tailed unpaired Student’s t test (B, D, and F–I) and one-way ANOVA (J).