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First published online 25 April 2006
doi: 10.1242/jcs.02942

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Regulation of pancreatic ß-cell insulin secretion by actin cytoskeleton remodelling: role of gelsolin and cooperation with the MAPK signalling pathway

¹ Department of Genetic Medicine and Development, University of Geneva Medical School, Switzerland
² Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY, USA

View larger version (56K): [in a new window]   Fig. 1. Confocal microscopy images showing the differences in the organisation and dynamic properties of the actin cytoskeleton in B1 and C3 cells. Response of cells to (A)10 minutes stimulation with 20 mM glucose after 0 mM glucose; (B) 16.7 mM after 2.8 mM glucose; and (C) 1 mM IBMX without glucose. F-actin was stained with phalloidin. Bars, 10 µm.

View larger version (26K): [in a new window]   Fig. 2. Effect of actin depolymerisation on the secretory response to glucose in B1 and C3 cells. (A) Latrunculin B-dependent depolymerisation of F-actin in B1 and C3 cells. Bar, 20 µm. (B) Stimulatory effect of actin depolymerisation by 10 µM latrunculin B on glucose-dependent insulin secretion in B1 and C3 cells. Left panels: basal (0 mM glucose for 1 hour) and stimulated (20 mM glucose for 1 hour) insulin secretion as a percentage of cell content; rights panels: fold-stimulation (*P<10-4). (C) Time-course of insulin secretion (20 mM glucose) by B1 and C3 cells with or without 10 µM latrunculin B. (D) Insulin content in B1 and C3 sublines with or without 10 µM latrunculin B treatment. Data in all cases are mean ± s.e.m. of three independent experiments.

View larger version (50K): [in a new window]   Fig. 3. (A) Western blot analysis of the differences in the level of gelsolin in B1, C3, mouse and rat primary pancreatic ß-cells. (B) Subcellular localisation of gelsolin (green) and F-actin (red) in B1 cells. No immunoreactivity was observed using control rabbit IgG (not shown). Bar, 10 µm.

View larger version (20K): [in a new window]   Fig. 4. Analysis of the role of gelsolin in insulin secretion by RNAi in B1 cells. (A) Western blot showing the effect of RNAi treatment on gelsolin protein levels in B1 cells. GFP-positive B1 cells transfected with either pSUPER-GFP gelsolin RNAi or pSUPER-GFP control plasmids were selected by FACS and used for immunoblotting. Equal loading was confirmed with an anti-actin antibody. (B,C) B1 cells were co-transfected with pSUPER-gelsolin RNAi or pSUPER-control RNAi plasmids and an hGH-expressing vector, and growth hormone secretion was measured after stimulation with 16.7 mM glucose (B), 1 mM IBMX (C, top panels), or a combination of 16.7 mM glucose and 1 mM IBMX (C, bottom panels). Data are mean ± s.e.m. of five independent experiments (B, right panel: *P<4x10-4). (D) Growth hormone secretion in B1 cells co-transfected with pSUPER-gelsolin RNAi or pSUPER-control RNAi plasmids and an hGH-expressing vector after a 2 hour pre-incubation with 10 µM latrunculin B. Data are mean ± s.e.m. of two independent experiments.

View larger version (46K): [in a new window]   Fig. 5. Analysis of the role of gelsolin in actin depolymerisation and insulin secretion by exogenous expression of an HA-tagged gelsolin cDNA construct in B1 and C3 cells. (A) Western blot showing the effect of exogenous expression of HA-gelsolin in B1 and C3 cells on the total level of gelsolin. Exogenous HA-gelsolin was specifically detected with an anti-HA-tag antibody (10 µg total protein/lane). (B) Immunofluorescence analysis of the subcellular localisation of HA-gelsolin (green) and the actin cytoskeleton (red) on B1 (top panels) and C3 cells (bottom panels) with or without 20 mM glucose stimulation. Nuclei (blue) were stained with DRAQ5TM. For glucose-stimulated B1 cells, two examples of HA-gelsolin-expressing cells are given and the actin (red) signal is boosted in the higher magnification image (zoom) to facilitate visualisation of actin/gelsolin co-localisation. Bars, 10 µm (unless stated otherwise). (C) Growth hormone secretion by B1 cells co-transfected with an HA-gelsolin expression plasmid or with a control empty plasmid. Data are mean ± s.e.m. of three replicates of a single experiment.

View larger version (24K): [in a new window]   Fig. 6. ERK1/2 activity contributes to the short-term glucose-induced insulin secretion in B1 cells. (A) Western blot analysis of the inhibitory effect of PD98059 on the activation (by phosphorylation) of ERK1/2 after 10 mM glucose stimulation in B1 cells. (B) Insulin secretion by B1 cells treated with PD98059 at three different time points. After 5 minutes glucose stimulation (top), B1 cells display a 60% decrease in their secretory response as compared to control DMSO-treated cells (*P<0.05). However, after 15 minutes stimulation (middle), the decrease in insulin secretion is reduced to 30% (**P<0.02), and is not present after 30 minutes glucose stimulation (bottom). Data in all cases are mean ± s.e.m. for basal and stimulated % insulin secretion (left panels) and secretion fold-stimulation (right panels) of three replicates of a single experiment. Stimulation conditions of 0 to 10 mM glucose were used to facilitate direct comparison with previously published work showing the effect of glucose-dependent ERK1/2 activation on insulin secretion (Longuet et al., 2005).

View larger version (99K): [in a new window]   Fig. 7. (A) Western blot analysis showing the differences in short-term glucose-induced ERK1/2 activation between B1 and C3 sublines and the stimulatory effect of latrunculin B pre-treatment on the short-term phosphorylation of ERK1/2. (B) Immunofluorescence analysis of the subcellular localisation of activated phosphoERK1/2 (green) and the actin cytoskeleton (red) on B1 (top panels) and C3 cells (bottom panels) before and after 5 minutes stimulation with 20 mM glucose. Nuclei (blue) were stained with DRAQ5TM. No immunoreactivity was observed using control rabbit IgG (not shown). Bars, 10 µm. (C) PhosphoERK1/2 accumulates at the tips of actin fibres in the vicinity of the plasma membrane (white arrows) after 20 mM glucose (left) and 16.7 mM glucose (right) stimulation in B1 cells. Bar, 10 µm. (D) Inhibition of ERK1/2 activation by PD98059 has no effect on the actin cytoskeleton remodelling properties of B1 or C3 cells as shown by confocal analysis of phalloidin-labelled cell samples + PD98059 before and after 5 minutes glucose stimulation. Bars, 10 µm.

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