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First published online May 4, 2004
doi: 10.1242/10.1242/jcs.01041

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Complexin I regulates glucose-induced secretion in pancreatic ß-cells

¹ Department of Internal Medicine, University of Lausanne, Lausanne, Switzerland
² Institute of Cellular Biology and Morphology, University of Lausanne, Lausanne, Switzerland

View larger version (37K): [in a new window]   Fig. 1. Complexin 1 (CPX I) is highly expressed in insulin-secreting cells. (a) Total RNA (15 µg) from different tissues were prepared and were subjected to northern blot analysis as described previously (Abderrahmani et al., 2001). RNAs were hybridized with a full-length rat cDNA CPX I probe that shares more than 80% sequence homology with CPX II (Takahashi et al., 1995). A transcript of 2.3 kb corresponding to CPX I is abundantly detected in the brain (used as a positive control for CPXs expression) and in three insulin-secreting cell lines (INS1, ßTC3 and MIN6 cells). A band of 5 kb corresponding to CPX II mRNA, is observed in the brain, in HeLa and RAW and at low levels in ßTC3 cells (Takahashi et al., 1995; McMahon et al., 1995). CPX II was not detected in INS1 and MIN6 cells. (b) Western blot analysis performed with total protein extracts from insulin-secreting cell lines and pancreatic islets confirmed expression of CPXs protein.

View larger version (45K): [in a new window]   Fig. 2. Immunoreactivity of CPXs is detected in insulin-positive cells from mouse islets. Immunohistochemistry for CPXs (a) (1/500 dilution) and for insulin (b) (1/500 dilution) was performed on isolated mouse islets. The overlay of the two images (c) shows that CPXs immunoreactivity is found in insulin-positive islet cells. The images were obtained by confocal microscopy.

View larger version (32K): [in a new window]   Fig. 3. The NRSE-like motif of the mouse complexin I gene binds REST. (a) EMSA with 32P-labeled CPXI-NRSE using nuclear extracts from HeLa cells. A slow migrating complex (arrow) is observed. This complex was disrupted by REST antibodies but not by unrelated antibodies against transcription factors OCT1, SP1 and CEBPß. (b) This DNA-binding activity was competed by adding a 100- or 600-fold molar excess of unlabeled wild-type NRSE of the MAPK8IP1 gene (WT) previously described to specifically bind REST (Abderrahmani et al., 2001). This binding activity was not disrupted by adding unlabeled mutated NRSE of the MAPK8IP1 gene (MUT). NE, nuclear extracts.

View larger version (28K): [in a new window]   Fig. 4. CPX I expression is efficiently silenced by plasmids directing the synthesis of small interfering RNAs (siRNA). Oligonucleotides were synthesized to specifically target CPX I and were cloned into pSUPER vector (see Material and Methods). The efficiency of siRNA was assessed by cotransfection of GFP-CPX I in the presence or in the absence of siRNA CPX I in INS1 cells. CPX I expression was detected using anti-GFP antibody. The western blot shows ectopic expression of CPX I, in the absence of siRNA CPX I (–) and in the presence of empty vector pSUPER (control) compared to untransfected cells (NT). However, CPX I expression is suppressed by adding siRNA CPX I.

View larger version (21K): [in a new window]   Fig. 5. The silencing of CPX I reduces the stimulus-induced secretion. The effect of siRNA CPX I on the secretory function in response to glucose and leucine was evaluated in (a) INS1 and (b) ßTC3 cells. Cells (5x105) were seeded in 24-well plates 1 day before transfection. Cells were transiently co-transfected with the plasmid encoding hGH as a reporter gene for secretion and with siRNA CPX I or pSUPER. Two days after transfection, the cells were cultured in 2 mM of glucose for 24 hours. Cells were then preincubated in KRBH for 60 minutes in 2 mM glucose (basal condition) and were subsequently incubated for 45 minutes in KRBH either under basal condition or with 20 mM glucose (plus 10 µM foskolin and 100 µM IBMX) or 20 mM leucine (plus 2 mM of glutamine). The amount of hGH released into the buffer and remaining inside the cells under basal and stimulatory conditions was determined by ELISA. Results were expressed as a percentage of hGH content. In the absence of siRNA CPX I, INS1 and ßTC3 cells were able to secrete hGH in response to glucose and leucine by 5-fold and 3-fold, respectively. By adding siRNA CPX I, cells were still able to secrete hGH in response to glucose and leucine compared to basal condition but the secretory response was significantly decreased by about 50% compared to cells transfected with pSUPER. (c) The 50% decrease of hGH secretion was also observed in response to 24 mM KCl in INS1 cells. (d) The decrease of stimulus-induced secretion by the silencing of CPX I occurs in a dose-dependent manner. The figure shows the mean ± s.e.m. of at least three independent experiments measured in triplicate (* P<0.01, ***P<0.001).

View larger version (16K): [in a new window]   Fig. 6. Differential effect of CXP I silencing on early and late secretory phases. INS-1 cells were transiently cotransfected with a plasmid encoding hGH and with the empty pSUPER vector and siRNA CPX I. After 3 days, the cells were incubated under basal condition (Basal) or in the presence of 20 mM glucose. After 10 minutes, the incubation medium was collected and replaced with fresh buffer for an additional 35-minute period. Cellular hGH content and the fraction of hGH released by the cells during the first 10 minutes of incubation (top panel) and during the successive 35 minutes (lower panel) were measured by ELISA. The figure shows the mean ± s.e.m. of at least three independent experiments measured in triplicate (*P<0.01, ***P<0.001).

View larger version (19K): [in a new window]   Fig. 7. Overexpression of CPX I reduces nutrient-induced secretory function. (a) Western blot analysis of CPX I overexpression in INS1 cells. The transient transfection of CMV-CPX I increased CPXI expression. The effect of overexpression of CPX I was assessed on the secretory ability of INS1 (b) and ßTC3 cells (c) in response to glucose and KCl. Cells were transiently co-transfected with the plasmid encoding CPX I (CMV-CPX I) or the empty vector (CMV) and with the plasmid encoding hGH vector. The release of hGH under basal and stimulatory (20 mM glucose supplemented by forskolin and IBMX or 24 mM of KCl) conditions was measured by ELISA. In both INS1 and ßTC3 cells, overexpression of CPX I significantly decreased, by 50%, the amount of secreted hGH in response to glucose and to KCl compared to cells transfected with CMV. The figure shows the mean ± s.e.m. of at least three independent experiments measured in triplicate (***P<0.001).