Phospholipase C-related catalytically inactive protein (PRIP) controls KIF5B-mediated insulin secretion

Abstract

We previously reported that phospholipase C-related catalytically inactive protein (PRIP)-knockout mice exhibited hyperinsulinemia. Here, we investigated the role of PRIP in insulin granule exocytosis using Prip-knockdown mouse insulinoma (MIN6) cells. Insulin release from Prip-knockdown MIN6 cells was higher than that from control cells, and Prip knockdown facilitated movement of GFP-phogrin-labeled insulin secretory vesicles. Double-immunofluorescent staining and density step-gradient analyses showed that the KIF5B motor protein co-localized with insulin vesicles in Prip-knockdown MIN6 cells. Knockdown of GABAA-receptor-associated protein (GABARAP), a microtubule-associated PRIP-binding partner, by Gabarap silencing in MIN6 cells reduced the co-localization of insulin vesicles with KIF5B and the movement of vesicles, resulting in decreased insulin secretion. However, the co-localization of KIF5B with microtubules was not altered in Prip- and Gabarap-knockdown cells. The presence of unbound GABARAP, freed either by an interference peptide or by Prip silencing, in MIN6 cells enhanced the co-localization of insulin vesicles with microtubules and promoted vesicle mobility. Taken together, these data demonstrate that PRIP and GABARAP function in a complex to regulate KIF5B-mediated insulin secretion, providing new insights into insulin exocytic mechanisms.

Keywords: GABARAP; Insulin secretion; KIF5B; PRIP; Vesicle transport.

Fig. 1.. Silencing of Prip1 and Prip2 in MIN6 cells promotes insulin secretion and vesicle movement.

(A) Prip knockdown in MIN6 cells. MIN6 cells were transfected with mixture of Prip siRNAs [Prip siRNA mix; Prip1-siRNAs (1, 2, 3) and Prip2-siRNAs (1, 2, 3)], cultured for 2 days, and then lysed. The whole cell lysates were processed for western blotting. Equivalent amounts of protein were loaded into each well (see β-tubulin staining). We obtained similar results from 3 different experiments, and a set of typical images is shown. (B) Enhanced insulin release from Prip-knockdown MIN6 cells. Prip-siRNA mix-transfected MIN6 cells were stimulated with 30 mM glucose, and the released insulin was measured every 1 min. Insulin secretion was normalized to intracellular insulin content, and is presented as a percentage of the total intracellular content. Values are presented as mean ± s.d. (n = 4). (C) Track plot recording of insulin vesicles in Prip-knockdown MIN6 cells. Cells were maintained in 5 mM glucose or stimulated with 30 mM glucose for 600 sec, and a time-lapse series of GFP-phogrin images was obtained every 5 sec for up to 690 sec. Particles of GFP-phogrin were selected at random, and each track, shown as a white line, was plotted based on the images. The dotted line shows a cell edge. We repeated these experiments 3 times, and analyzed approximately 70 displaced vesicles. A representative set of images is shown. (D) Analysis of vesicle mobility using GFP-phogrin-labeled vesicles. The data from 3 experiments were combined, and the percentage of vesicles travelling at a velocity greater than 10 µm per min is shown. (E) The average accumulated distance per vesicle was calculated. Each relative value is based on the distance traveled by control cells (without Prip-siRNA transfection) under low-glucose conditions. Values are presented as mean ± s.e. (from the left in each indicated condition; n = 70, 78, 81, and 63, respectively). *p<0.05, ^§§p<0.01, ***p<0.001, ^†††p<0.001; n.s., not statistically significant. Scale bar: 5 µm.

Fig. 2.. KIF5B-mediated insulin vesicle transport is regulated by PRIP.

(A) Insulin secretion analysis of kif5b-knockdown MIN6 cells. MIN6 cells transfected with Kif5b-siRNA (1) and Prip-siRNA mix (+) or scrambled siRNA (−) were stimulated with 30 mM glucose. Released insulin was collected and measured every 1 min. The mean percentage of total insulin content is shown. Values are presented as mean ± s.d. (n = 3). (B) The average accumulated distance per vesicle was calculated. Each value is the average distance travelled by the cells relative to that of cells transfected with scrambled siRNA under high glucose (30 mM) conditions. Values are presented as mean ± s.e. (from the left in each experiment; n = 120, 116, and 56, respectively). (C–E) Co-localization analyses of KIF5 (green) and β-tubulin (red) (C), KIF5 (green) and insulin (red) (D), and β-tubulin (green) and insulin (red) (E) in MIN6 cells transfected with scrambled siRNA (upper panels) or PRIP1-siRNAs (1, 2, 3) and PRIP2-siRNAs (1, 2, 3) (Prip-siRNA mix; lower panels). Cells were stimulated with 30 mM glucose for 10 min, fixed with 3.7% paraformaldehyde, subjected to immunocytochemistry with a specific antibody, and processed for confocal microscopy. The yellow and blue pseudo-colors in the PDM images show areas of high and low co-localization, respectively. Magnified images of panels C–E are shown in supplementary material Fig. S8A–C. The dotted line shows a cell edge. A set of typical images from 3 independent experiments is shown. (F–H) Statistical analysis of the co-localization experiments in panels C–E. Overlap coefficients were calculated in each experiment. Values are presented as mean ± s.d. (from the left in each graph, n = 60 and 45 (F); 96 and 104 (G); 84 and 60 (H), respectively). *p<0.05, ***p<0.001, ^††p<0.01, ^†††p<0.001; n.s., not statistically significant. Scale bars: 5 µm.

Fig. 3.. Knockdown of Prip increases the amount of KIF5 in the secretory vesicle fraction.

(A–D) Cells were transfected with scrambled siRNA (A,B) or both Prip1-siRNAs (1, 2, 3) and Prip2-siRNAs (1, 2, 3) (Prip siRNA mix; C,D). The cells were stimulated with (B,D) or without (A,C) 30 mM glucose for 10 min. Equivalent protein in the cell homogenates was fractionated in OptiPrep™ discontinuous gradients (3%, 7.5%, 18%, and 35%). The fractions were analyzed by western blotting using the indicated specific antibodies. We performed each experiment 3 times, and a set of typical images is shown. (E,F) The vesicle fraction (fraction 11), which was defined as the fraction with high levels of Rab27a, phogrin, and insulin, was collected, and KIF5 content was analyzed by western blotting. The total amounts of KIF5 in Prip-knockdown (+) and scrambled siRNA-transfected cells (−) did not differ (lowest blot in panel E). A set of typical images is shown. The calculated mean value for each band is shown in the graph (F). Values are presented as mean ± s.d. (n = 3); *p<0.05, ^†p<0.05.

Fig. 4.. GABARAP enhances insulin vesicle transport by regulating insulin vesicle localization with KIF5 and β-tubulin, which is negatively regulated by PRIP.

(A) Insulin secretion from Gabarap-knockdown MIN6 cells. Gabarap-siRNA-transfected MIN6 cells co-transfected with or without Prip1-siRNAs (1, 2, 3) and Prip2-siRNAs (1, 2, 3) (Prip siRNA mix) were cultured for 2 days. The cells were stimulated with 30 mM glucose, and the released insulin was measured every 1 min. The mean value for the percentage of total insulin content is shown. Values are presented as mean ± s.d. (n = 3). (B) The mean accumulated distance per vesicle was calculated under high-glucose conditions. Each value is presented relative to the distance traveled by the control (black bar). Values are presented as mean ± s.e. (from the left, n = 47, 168, 112, 45, 169, and 66). (C–J) Co-localization analyses of KIF5 (green) and β-tubulin (red) (C), KIF5 (green) and insulin (red) (D), and β-tubulin (green) and insulin (red) (E) in MIN6 cells transfected with scrambled siRNA (upper panels) or Gabarap-siRNA (1) (lower panels), or KIF5 and insulin (I) in MIN6 cells transfected with an empty vector (upper panels) or myc-tagged GABARAP2–35 (lower panels) after 30 mM glucose stimulation for 10 min. Cells were fixed with 3.7% paraformaldehyde, subjected to immunocytochemistry with each specific antibody, and processed for confocal microscopy. The yellow and blue pseudo-colors in the PDM images show areas of high and low co-localization, respectively. Magnified images of panels C–E are shown in supplementary material Fig. S8A–C. The myc-expressing cells (the left panel of each experiment) isolated with a FITC-conjugated anti-myc antibody were analyzed (I), and the arrowheads and arrows in the magnified images represent the areas of co-localization and non-colocalization between KIF5 (obtained blue images were replaced with green) and insulin (red), respectively. The single-color images are shown in supplementary material Fig. S9A. The dotted line shows a cell edge. A set of typical images from more than 81 cells in 3 independent experiments is shown. The overlap coefficient was calculated and is shown in panels F–H and J, and the values are presented as mean ± s.d. (from the left in each graph, n = 96 and 100; 128 and 124; 124 and 100; 93 and 81, respectively). *p<0.05, ***p<0.001, ^†p<0.05, ^†††p<0.001; n.s., not statistically significant. Scale bars: 5 µm.

Fig. 5.. Prip silencing enhances the co-localization of GABARAP with insulin vesicles and β-tubulin.

Co-localization of GABARAP (green) with insulin (red) (A) or β-tubulin (red) (B) in MIN6 cells transfected with scrambled siRNA (upper panels) or Prip siRNA mix [Prip1-siRNAs (1, 2, 3) and Prip2-siRNAs (1, 2, 3), lower panels] after stimulation with 30 mM glucose for 10 min. Magnified images of panels A and B are shown in supplementary material Fig. S9B,C. A set of representative images from 3 independent experiments is shown. The overlap coefficient was calculated. Values are presented as mean ± s.d. (from left in each graph of panels A and B; n = 144 and 112; 84 and 160, respectively); ***p<0.001. Scale bars: 5µm.

Fig. 6.. Overexpression of GABARAP40–67 promotes the co-localization of GABARAP with insulin vesicles, microtubules, and KIF5, and facilitates insulin vesicle movements.

(A) Quantitative analysis of insulin vesicle movement in MIN6 cells transfected with or without pIRES2-DsRed/GABARAP40–67. The accumulated distance of a randomly selected track of insulin vesicles was calculated. Values are presented as mean ± s.e. (from the left, n = 214, 233, and 326, respectively). (B–F) Co-localization analyses of GABARAP (green) and insulin (blue) (B), GABARAP (green) and β-tubulin (blue) (C), β-tubulin (green) and insulin (blue) (D), KIF5 (green) and β-tubulin (blue) (E), and KIF5 (green) and insulin (blue) (F) in MIN6 cells transfected with pIRES2-DsRed/empty (upper panels) or pIRES2-DsRed/GABARAP40–67 (lower panels) in response to stimulation with 30 mM glucose for 10 min. Endogenous GABARAP was stained with an anti-GABARAP antibody, which recognized amino acid residues 1–39 of GABARAP. Magnified images of B–F are shown in supplementary material Fig. S10A–E. A set of typical images from 3 independent experiments is shown. The dotted line shows a cell edge. The overlap coefficient in each co-localization experiment was calculated. Values are presented as mean ± s.d. [from the left in each graph, n = 64 and 76 (B); 40 and 40 (C); 40 and 40 (D); 52 and 60 (E); and 40 and 60 (F)]. *p<0.05, ***p<0.001, ^†††p<0.001. Scale bars: 5 µm.

Fig. 7.. Dissociation of GABARAP from PRIP in response to glucose stimulation.

MIN6 cells transfected with myc-tagged GABARAP were stimulated with low (5 mM) or high glucose (30 mM), followed by immunoprecipitation with an anti-myc antibody or control IgG. Immunoprecipitates were analyzed by SDS-PAGE and western blotting using the indicated primary antibodies. Similar results were obtained from 3 independent experiments.

Fig. 8.. PRIP deficiency increases glucose-induced insulin secretion and exocytic events in pancreatic islets.

(A) Time course of glucose-stimulated insulin secretion from Prip-DKO and wild-type mouse pancreatic islets. Isolated pancreatic islets were stimulated with 20 mM glucose, and the amount of insulin released was measured. Insulin secretion was normalized to intracellular insulin content, and is presented as a percentage of the total intracellular content. Insulin secretion per min was compared during the indicated periods. The first and second phases were defined as 2–7 min, and over 7 min, respectively. Values are presented as mean ± s.e. (n = 5 for each genotype). (B) Time course of exocytic events observed by two-photon microscopy during glucose stimulation (20 mM) in pancreatic islets from Prip-DKO and wild-type mice. More than 400 exocytic events from >100 cells of each genotype in 5 independent experiments were analyzed. The number of exocytic events is expressed as events per cell per 30-sec interval and is shown as the mean number of events during the indicated time periods. Values are presented as mean ± s.e. (wild type, n = 126; Prip-DKO, n = 116). *p<0.05, **p<0.01.