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Endocrinology, doi:10.1210/en.2007-0974
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Endocrinology Vol. 149, No. 5 2494-2505
Copyright © 2008 by The Endocrine Society

Dual Effect of Cell-Cell Contact Disruption on Cytosolic Calcium and Insulin Secretion

Fabienne Jaques, Hélène Jousset1, Alejandra Tomas1, Anne-Lise Prost, Claes B. Wollheim, Jean-Claude Irminger, Nicolas Demaurex and Philippe A. Halban

Department of Genetic Medicine and Development (F.J., A.T., A.-L.P., J.-C.I., P.A.H.) and Department of Cell Physiology and Metabolism (H.J., C.B.W., N.D.), University of Geneva Medical Center, 1211 Geneva-4, Switzerland

Address all correspondence and requests for reprints to: Fabienne Jaques, Department of Genetic Medicine and Development, University of Geneva Medical Center, 1211 Geneva-4, Switzerland. E-mail: fabienne.jaques{at}medecine.unige.ch.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cell-to-cell interactions play an important role in insulin secretion. Compared with intact islets, dispersed pancreatic β-cells show increased basal and decreased glucose-stimulated insulin secretion. In this study, we used mouse MIN6B1 cells to investigate the mechanisms that control insulin secretion when cells are in contact with each other or not. RNAi-mediated silencing of the adhesion molecule E-cadherin in confluent cells reduced glucose-stimulated secretion to the levels observed in isolated cells but had no impact on basal secretion. Dispersed cells presented high cytosolic Ca2+ activity, depolymerized cytoskeleton and ERK1/2 activation in low glucose conditions. Both the increased basal secretion and the spontaneous Ca2+ activity were corrected by transient removal of Ca2+ or prolonged incubation of cells in low glucose, a procedure that restored the ability of dispersed cells to respond to glucose (11-fold stimulation). In conclusion, we show that dispersed pancreatic β-cells can respond robustly to glucose once their elevated basal secretion has been corrected. The increased basal insulin secretion of dispersed cells is due to spontaneous Ca2+ transients that activate downstream Ca2+ effectors, whereas engagement of cell adhesion molecules including E-cadherin contributes to the greater secretory response to glucose seen in cells with normal intercellular contacts.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PANCREATIC β-CELLS control blood glucose homeostasis by their capacity to secrete insulin in response to metabolic needs. Defects in regulation of insulin secretion lead to severe metabolic problems, including diabetes, as an inevitable consequence.

The endocrine pancreas is organized as discrete clusters of cells, the islets of Langerhans that are composed in large part of {alpha}-cells, β-cells, {delta}-cells, and PP-cells, with β-cells being the main component (1, 2). It was well established more than 20 yr ago that the integrated secretory response of intact islets is greater than that of dispersed islet cells, suggesting that intraislet interactions are necessary for a normal secretory response (3, 4, 5, 6). At least part of this effect is due to homotypic β-cell-to-β-cell interactions, as confirmed in vitro by the use of either rat primary β-cells or transformed mouse (MIN6) β-cells. When islet cells are dispersed, they show an increased basal insulin secretion and a decreased glucose-stimulated insulin secretion (GSIS) (3, 5, 7, 8), compared with intact islets. Both secretory defects are reversed by reaggregation of islet cells (3, 7, 9), indicating that cell-cell communication is essential to provide low insulin release in periods of starvation and sufficient amounts of insulin after food intake. At present, however, only a few elements are known that explain how cell-cell contacts affect basal secretion as well as GSIS.

Glucose stimulation of β-cells provokes a cascade of events including an elevation of the ATP to ADP ratio, cell membrane depolarization, intracellular free calcium concentration ([Ca2+]i) elevation, and finally exocytosis of insulin containing granules. Nutrient-induced increases in intracellular free Ca2+ concentrations are thus a key trigger for insulin release from pancreatic islet β-cells. Indeed, Ca2+ activates directly proteins involved in fusion events including soluble N-ethylmaleimide sensitive fusion factor attachment protein receptor proteins, protein kinases like protein kinase A and protein kinase C, and Ca2+-sensitive proteins like gelsolin or scinderin that are involved in essential cytoskeleton remodeling needed for exocytosis (10, 11, 12).

In this study we used the highly differentiated transformed mouse β-cell MIN6 subclone B1 (13) to investigate the intracellular mechanisms that link cell-cell contact to insulin secretion. We show that isolated cells have a weak insulin secretory response to glucose and abnormally elevated basal insulin secretion. We further demonstrate that these two defects have different origins. Engagement of the Ca2+-dependent adhesion molecule E-cadherin between cells in contact with one another is partially responsible for the normal response to glucose, whereas increased Ca2+-influx underlies the elevated basal secretion from dispersed cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
MIN6B1 cells were cultured in DMEM supplemented with 15% fetal calf serum, 25 mM glucose, 71 µM 2-β-mercaptoethanol, 2 mM glutamine, 100 U/ml penicillin, and 100 mg/liter streptomycin. For the dispersed or confluent conditions, 105 cells were attached either in the center (confluent, 105 cells per 4 mm2) or all over the surface (dispersed, 105 cells per 200 mm2) of 24-well plates, respectively. For the confluent condition, cells were first plated as a droplet of 18 µl of medium containing 105 cells; after 1 min of attachment, 482 µl of culture medium was added delicately. For the dispersed condition, 105 cells were seeded directly into wells in 500 µl of medium.

Insulin secretion assays
Cells were washed three times with a modified Krebs-Ringer bicarbonate HEPES buffer [KRBH; 125 mM NaCl, 4.74 mM KCl, 1 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 5 mM NaHCO3, 25 mM HEPES (pH 7.4), and 0.1% BSA] supplemented with 2.8 mM glucose and preincubated for 2 h in this same buffer. Cells were then incubated for 1 h at 37 C in KRBH 2.8 mM glucose (basal secretion), followed by 1 h at 37 C in KRBH 16.7 mM glucose or secretagogues of interest (stimulated secretion). The only exception was stimulation with KCl, for which the incubation period was limited to 10 min. To block voltage-dependent calcium channels (VDCC) channels, 3 or 50 µM of nifedipine were added to the buffer during the preincubation and incubation periods. For the secretion tests with a longer basal incubation, the basal preincubation of 2 h was followed by three successive 90-min incubations in basal conditions (KRBH 2.8 mM glucose, 37 C) followed by a 90-min incubation in stimulated conditions (KRBH 16.7 mM glucose, 37 C). The secretion media (basal and stimulated) were centrifuged at 2000 rpm for 5 min to remove any cells that may have detached from the well during incubations. The cells were then extracted in acid ethanol and insulin was measured in the incubation buffers and cellular extracts by RIA using the charcoal separation technique (14). Rat insulin was used as a standard with a guinea pig antiporcine insulin antibody and 125I-insulin as a tracer. Secretion is expressed as a percentage of total content (cell extract + basal and stimulated secretion).

Detection of apoptosis
Cells were seeded in 24-well plates (dispersed or confluent) 48 h before apoptosis measurement. Apoptosis was quantified using the cell death detection ELISAPLUS kit (Roche, Basel, Switzerland), which detects mono- and oligonucleosomes present in the cytoplasm of apoptotic cells, according to the manufacturer’s instructions. In parallel, cells were trypsinized and counted.

Pulse chase
Cells were washed three times with KRBH 2.8 mM glucose before being preincubated 1 h at 37 C in the same buffer. Cells were then incubated for 30 min at 37 C in KRBH 2.8 mM glucose containing 1 mCi/ml [3H]leucine (specific radioactivity, 150 Ci/mmol; Anawa Trading, Zurich, Switzerland). The cells were then washed three times with KRBH 2.8 mM glucose and incubated for 90 min at 37 C with KRBH 2.8 mM glucose (basal incubation) followed by 1 h at 37 C with KRBH 16.7 mM glucose (stimulated incubation). The two secretion media (basal and stimulated) were centrifuged at 2000 rpm for 5 min to remove any cells that may have detached from the dish during incubations and acidified before analysis. The cells were extracted in 1 M acetic acid and 0.1% BSA. Samples were analyzed by reverse-phase HPLC using a well-established method allowing for separation and quantification of radiolabeled proinsulin, conversion intermediates, insulin, and C-peptide as described previously (15, 16).

RNA isolation
Cells in dispersed or confluent conditions were lysed for preparation of total RNA, using the RNeasy minikit (QIAGEN, Basel, Switzerland) according to the manufacturer’s instructions. The quality of the starting RNA was tested (2100 bioanalyzer, RNA 6000 Pico LabChip; Agilent, Palo Alto, CA) and used only if satisfactory. Criteria were the absence of degradation of the ribosomal RNA that represents 90–95% of total RNA and a ratio 28S:18S equal to 1.8–2.0. cRNA quality was also tested after both amplifications using RNA 6000 Nano LabChip.

Microarray hybridization
Hybridization targets were obtained after a double-amplification procedure according to the protocol developed by Affymetrix (GeneChip eukaryotic small sample target labeling assay version II; Affymetrix, Santa Clara, CA). A hybridization mixture containing 17.5 µg of biotinylated cRNA was generated and the biotinylated cRNA was hybridized to Affymetrix GeneChip MOE 430A containing approximately 22,000 genes. Three chips were hybridized for each condition, using RNA from three independent experiments. Chips were visualized on an Scs 3000 gene scanner (Affymetrix) and image files analyzed. Fold differences were calculated as the ratio between the average values within each condition. Signal values and detection calls (present or absent) for all samples were determined by using Affymetrix Microarray 5.0. Transcripts with 2-fold and greater difference between groups and a P < 0.05 were considered significant.

RNA interference (RNAi)-mediated silencing of endogenous E-cadherin
A 64-bp sequence encoding 21-bp-long small hairpin RNAs (shRNAs) specific for mouse E-cadherin was cloned into both pSUPER and pSUPER-GFP plasmids (OligoEngine, Inc., Seattle, WA). A similar sequence encoding a nonspecific shRNA without mammalian homology was used as negative control. The shRNA sequence was tested for its capacity to knock down E-cadherin in MIN6B1 cells as follows. Cells were transfected using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) with the pSUPER-GFP plasmid containing either the shRNA against E-cadherin or the negative control. Cells were cultured for 72 h for RNAi expression before selection of green fluorescent protein-positive cells by fluorescence-activated cell sorting. Selected cells were used for Western blot or RT-PCR analysis of E-cadherin protein levels, compared with negative control cells. The shRNA sequence 5'-AAC CCA GAG CTG CTC ATG TTT-3' exhibited a good silencing capacity and was used in all subsequent experiments.

GH secretion assay
To study secretion specifically from transfected cells, cells were cotransfected with a human GH (hGH)-expressing vector and the vector of interest (1:3 DNA ratio). One day after transfection, cells were trypsinized and plated in dispersed or confluent conditions as described above. Hormone secretion was measured 48 h later (72 h after the transfection) as described above. The amount of hGH (used as a surrogate marker for insulin secretion from transfected cells) in the incubation buffers and cell extracts was measured by ELISA using the hGH ELISA kit from Roche Diagnostics (Basel, Switzerland), following the manufacturer’s instructions. Secretion of hGH is expressed as a percentage of the hGH content (cell extract + basal and stimulated secretion).

Cytosolic Ca2+ measurements
Ca2+ measurements were performed in KRBH buffer as described for the secretion test, using a previously described method (17). Cells were loaded for 30 min with 2 µM fura-2/AM at room temperature in the dark, washed twice, and equilibrated for 10 min to allow deesterification. To monitor [Ca2+]i, cells were alternatively excited at 340 and 380 nm with a monochromator (DeltaRam; Photon Technology International Inc., Monmouth Junction, NJ) through a 430 DCLP dichroic mirror, and emission was monitored through a 510WB40 filter (Omega Optical, Brattleboro, VT). Fluorescence emission was imaged using a cooled, 16-bit, charge-coupled device, back-illuminated, frame transfer Micro-Max camera (Princeton Instruments, Roper Scientific, Trenton, NJ). Image acquisition and analysis were performed with the MetaFluor 6.2 software (Universal Imaging, Westchester, PA).

Fluorescence microscopy
Cells grown 48 h on 35-mm glass-bottom microwell dishes (MatTek, Ashland, MA) coated with poly-L-lysine were washed in PBS, fixed at room temperature for 20 min in 4% paraformaldehyde, washed three times with PBS, and permeabilized at room temperature for 4 min in PBS + 0.5% Triton X-100. Cells were then washed three times in PBS and blocked for 30 min with PBS + 0.5% BSA. For actin labeling, cells were incubated with Alexa Fluor 546-phalloidin (Invitrogen, Basel, Switzerland) for 20 min after fixation and permeabilization. Nuclei were detected with the DNA-binding dye DRAQ5 (Biostatus Ltd., Shepshed, UK). Labeled cells were washed three times, and samples were kept in PBS at 4 C before their observation under a Zeiss LSM 510 inverted confocal microscope (Carl Zeiss AG, Jena, Germany) using a x63 oil immersion lens. The lowest plane from the side of attachment was selected to visualize cortical actin at the basal membrane level. Images were acquired and processed using the Lsm510 software (Carl Zeiss).

Western blot analysis
To analyze ERK protein phosphorylation and quantify E-cadherin level in transfected cells, cells were washed with ice-cold PBS without Ca2+/Mg2+ supplemented with 1 mmol/liter sodium vanadate and then lysed in sample buffer 1x [62 mM/liter Tris-Cl (pH 6.8), 2% sodium dodecyl sulfate, 5% glycerol, and 1% 2-mercaptoethanol]. Protein concentrations were determined with the amido black method (18), and equal amounts of total protein were loaded for SDS-PAGE. All samples, after separation on an SDS-PAGE gel, were electroblotted onto nitrocellulose membranes (Schleicher & Schuell, Bassel, Germany) for immunoblotting with the appropriate antibody: polyclonal anti-phospho-ERK1/2 (Thr-202/Tyr-204) and anti-ERK1/2 from Cell Signaling Technology-Bioconcept (Allschwil, Switzerland); rabbit polyclonal anti-E-cadherin [prepared as previously described (19)]; monoclonal antiactin from Chemicon International (Temecula, CA); and antimouse and antirabbit horseradish peroxidase from Amersham Pharmacia Biotech (Dübendorf, Switzerland). A Kodak image station (Rochester, NY) was used for visualization of the bands.

Statistics
Data are presented as mean ± SE for n independent experiments. Significance of differences was assessed by Student’s two-tailed t test for unpaired groups.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Insulin secretion from confluent and dispersed pancreatic β-cells
We showed previously that the MIN6 subclone B1 responds better to glucose when cells are confluent (13), similarly to primary pancreatic β-cells (7).

To further characterize this phenotype, we standardized the culture conditions to obtain confluent or dispersed cells cultured in the same volume of medium. To this end, the same number of cells (105) was plated either as a droplet covering 4 mm2 (confluent) or all over the 200 mm2 surface of the well (dispersed). After 48 h in culture, confluent cells were nicely spread, whereas dispersed cells remained round (Fig. 1AGo). As previously shown, confluent cells displayed robust insulin secretion when exposed to 16.7 mM glucose, whereas only a weak response was observed in dispersed cells (22- vs. 1.7-fold stimulation, Fig. 1BGo). The lack of response to high glucose in dispersed cells was due to the combination of elevated basal secretion (3.8% of total insulin in dispersed cells vs. 0.45% in confluent cells, P < 0.001) and decreased stimulated secretion (6.5% of total insulin in dispersed cells vs. 9.8% in confluent cells, P < 0.001) (Fig. 1BGo). The elevated basal secretion in dispersed cells was not due to hypersensitivity to glucose because secretion tests performed with no glucose (0 mM) or with less glucose (1.4 mM) instead of normal basal conditions (2.8 mM) showed the same results with basal insulin secretion still elevated (3.7 and 3.6%, respectively, of total insulin per hour).


Figure 1
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  		FIG. 1. Spreading and insulin secretion of dispersed vs. confluent cells. A, Phase-contrast microscopy of living dispersed or confluent cells. After 48 h in culture, dispersed cells (105 cells per 200 mm2) do not spread and remain round, whereas confluent cells (105 cells per 4 mm2) spread and form numerous cell-cell contacts. B, Insulin secretion in response to glucose. Dispersed or confluent cells were seeded in 24-well plates, cultured for 2 d, and preincubated under basal conditions for 2 h, followed by 1 h each at 2.8 mM (basal, {square}) and 16.7 mM (stimulated, {blacksquare}) glucose. The supernatant was recovered for insulin measurement. Results are expressed as a percentage of total insulin (cell content + secreted). Data are mean ± SEM of at least three independent experiments. *, P < 0.001 vs. dispersed cells at the same glucose concentration.

 
Other studies have shown that confluent cells have a higher insulin content than dispersed cells (20, 21). In our model system, there was no significant difference in cellular insulin content between confluent or dispersed cells (3.3 ± 0.65 µg insulin per 106 cells in confluent vs. 3.1 ± 0.5 µg insulin per 106 cells in dispersed cells, n = 5). The level of insulin mRNA was also quantified in both conditions by real-time PCR, and the same amount was found in confluent and dispersed cells (ratio mRNA confluent cells to mRNA dispersed cells: 0.89 ± 0.2, n = 4). Altered secretion from dispersed vs. confluent cells was therefore not the reflection of altered insulin production or storage. It has been suggested that elevated basal insulin secretion could reflect poor survival in culture of dispersed pancreatic β-cells. We therefore compared the number of apoptotic cells in the dispersed and confluent conditions and found no differences between these two situations (ratio apoptotic cells for dispersed vs. confluent = 1.07 ± 0.44, n = 3). Finally, cell proliferation was measured by 5-bromo-2'-deoxyuridine incorporation. No significant difference was found between dispersed and confluent cells after 48 h in culture, at either high glucose or after low glucose incubation (not shown).

Microarray gene analysis
Contacts between β-cells could modulate expression of genes that may predispose cells to become glucose responsive and competent for well regulated insulin secretion. To evaluate this, we performed high-density microarray experiments (Affymetrix) to identify genes differentially expressed in dispersed and confluent cells. To this end, mRNA was analyzed from dispersed or confluent cells after 48 h in standard culture conditions. No significant differences (considered as >2-fold, P < 0.05 for n = 3 independent experiments) were found using these experimental and analytical conditions.

Effect of E-cadherin down-regulation on cell spreading and insulin secretion
To study more directly the role of cell-cell interactions in both components of insulin secretion (basal and stimulated), we decided to down-regulate E-cadherin, one of the most important adhesion molecules in pancreatic β-cells (22, 23) that plays a major role in insulin secretion (24). We found that E-cadherin was expressed at identical levels in dispersed and confluent cells by microarray analysis and by quantitative real-time RT-PCR (ratio E-cadherin mRNA confluent cells to mRNA isolated cells: 1.07 ± 0.04, n = 3). We also checked the E-cadherin protein levels by Western blot analysis and found no difference between isolated and confluent cells (not shown). However, because E-cadherin needs homotypic association to be activated, it remains possible that engagement of E-cadherin is responsible for the secretory signature of confluent cells. To test this directly, E-cadherin expression was decreased by transfection with shRNA (Fig. 2AGo). Densitometric analysis of immunoblots using extracts of transfected cells demonstrated a 44% reduction in E-cadherin protein content (Fig. 2BGo, right panel), consistent with the approximately 50% reduction in E-cadherin mRNA expression in cells expressing RNAi for E-cadherin (Fig. 2BGo, left panel). hGH was used as a surrogate marker for insulin secretion from the subpopulation of transfected cells because this hormone is stored (25) and cosecreted with insulin in pancreatic β-cells (26, 27, 28). Control experiments were performed with cells cotransfected with hGH and the pSUPER plasmid containing a control RNAi with no mammalian homology (Fig. 2Go, RNAi CTRL). No difference in secretion was observed in dispersed cells transfected with E-cadherin RNAi (Fig. 2CGo). By contrast, down-regulation of E-cadherin in confluent cells was associated with a decrease in stimulated release of hGH (from 40 to 23.5%), but was without effect on basal secretion (Fig. 2CGo). These results show that E-cadherin plays a role in secretion when confluent cells are stimulated with glucose but that decreasing the amount of this cell adhesion molecule in confluent cells by approximately 50% does not impact on basal secretion.


Figure 2
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  		FIG. 2. Effect of E-cadherin down-regulation on insulin secretion. A, Western blot for E-cadherin (ECAD). Experiment was performed on cells transfected with pSUPER-GFP containing either RNAi control (RNAi CTRL) or RNAi for E-cadherin and then sorted by fluorescence-activated cell sorting. Actin was used as a reference. B, Quantification of E-cadherin reduction in mRNA expression and protein content. Mean ± SEM, n = 3 independent experiments, for data normalized to levels of cells with RNAi CTRL. *, P < 0.05 vs. RNAi control. C, Cells were cotransfected with hGH and pSUPER containing either RNAi control or RNAi for E-cadherin. hGH was used as a marker to monitor the secretory potential of transfected cells with decreased E-cadherin expression. Data were from one experiment performed in quadruplicate and representative of three independent experiments. *, P < 0.003 vs. RNAi control.

 
Trafficking of proinsulin in confluent vs. isolated cells
In contrast to the regulated secretory pathway that needs a stimulus for exocytosis, in the constitutive pathway, proteins are synthesized and continuously secreted (29). In pancreatic β-cells, both the regulated and constitutive secretory pathway are known to exist, but these cells normally sort more than 99% of newly synthesized proinsulin to secretory granules for regulated release (30). The elevated basal secretion of isolated cells persisted in the complete absence of glucose and could thus reflect abnormal constitutive secretion of proinsulin rather than regulated insulin release because the antibodies used in conventional insulin RIA are not able to differentiate between insulin and proinsulin. To investigate this hypothesis, the secretion of newly synthesized proinsulin and insulin was followed by pulse chase. Using this protocol, the rapid secretion of (unprocessed) proinsulin for the first 90 min of a postlabel (chase) incubation under basal conditions is considered to be due to release via the constitutive secretory pathway (31). We found no significant difference in proinsulin to insulin conversion in dispersed vs. confluent cells (proinsulin conversion at end of chase period = 94.3 ± 1 vs. 92.6 ± 1.6%, respectively, n = 3). Furthermore, and more specifically, the percentage of intact, nonconverted labeled proinsulin secreted during the 90-min basal chase period was not significantly different between the two conditions (1.4 ± 0.8% in dispersed vs. 0.7 ± 0.3% in confluent cells, n = 3). This indicates that elevated basal secretion in dispersed cells is not due to elevated constitutive release of freshly produced proinsulin. These data incidentally confirm that MIN6B1 cells are highly differentiated and like primary β-cells are able to sort about 99% of newly synthesized proinsulin to the regulated secretory pathway.

This result was confirmed by using cycloheximide during the secretion test to block protein synthesis and thereby avoid release of freshly synthesized proinsulin. Because basal secretion from dispersed cells remained elevated under these conditions (similar to control condition), we confirmed that elevated basal insulin release in isolated cells is due to abnormal insulin secretion via the regulated pathway.

Effect of different secretagogues on insulin secretion
To further characterize insulin secretion in dispersed cells, compared with confluent cells, seven secretagogues were used that activate different and specific steps of the cascade(s) of events leading to insulin secretion [20 mM leucine, 20 mM arginine, 30 mM KCl, 100 nM glibenclamide, 1 mM 3-isobutyl-1-methylxanthine (IBMX), 100 nM phorbol-12-myristate-13-acetate (PMA), and 10 µM mastoparan]. Confluent cells responded to different degrees to all secretagogues tested (see Table 2Go). Dispersed cells responded to 3-isobutyl-1-methylxanthine (3.3-fold), PMA (15-fold), and mastoparan (4.4-fold) but failed to respond with a statistically significant increase in insulin secretion to leucine, arginine, KCl, or glibenclamide (Table 1Go). However, likely as a consequence of the very high levels of basal secretion from dispersed cells, the absolute amounts of insulin secreted in presence of these secretagogues in fact exceeded that from the confluent cells to these same stimuli (Tables 1Go and 2Go). We suggest that such constitutively high basal secretion could mask a possible modest increase after exposure to these particular secretagogues. Nevertheless, dispersed cells thus failed to respond to secretagogues that are dependent on changes in cytosolic Ca2+ but did respond to secretagogues that act independently of Ca2+ changes as long as [Ca2+]i exceeds a certain threshold (32).


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  		TABLE 2. Insulin secretion from confluent cells in response to various secretagogues

 

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  		TABLE 1. Insulin secretion from dispersed cells in response to various secretagogues

 
Cytosolic Ca2+ measurement
The previous data on insulin secretion strongly suggested that dispersed cells have constitutively elevated levels of cytosolic Ca2+. To verify this hypothesis, we investigated the impact of cell-cell contacts on cytosolic Ca2+ homeostasis. For this purpose, cells were loaded with fura2/AM and cytosolic Ca2+ was recorded during 20 min in low glucose. As shown in Fig. 3Go, confluent cells had stable Ca2+ levels, whereas dispersed cells presented spontaneous [Ca2+]i elevations in low glucose. The Ca2+ elevations were transient and of variable amplitude and were considered significant only when they exceeded by more than 1 SD the averaged maximal [Ca2+]i values measured in the population of confluent cells during the same 20-min period (Fig. 3EGo, gray bar). Using this criterion, a substantial fraction of dispersed cells were considered as active (28%), compared with only 3% of confluent cells (Fig. 3Go, P < 0.0001).


Figure 3
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  		FIG. 3. Effect of cell-cell contact disruption on Ca2+ homeostasis. MIN6B1 cells were loaded with the calcium-sensitive indicator fura2-AM after a 2-h incubation at low glucose to match the secretion assays (Fig. 1Go). A and B, fura2 ratio images of confluent (A) and dispersed cells (B) represented in intensity modulated display mode. C and D, Representative Ca2+ recordings of confluent (C) and dispersed cells (D) incubated at 2.8 mM glucose for 20 min. Fura2 ratios were normalized to baseline value (R/Ro). E, Scatter graph of the maximal ratio values recorded during the 20-min period in confluent and dispersed cells. Each point represents a single-cell measurement with n = 102 for confluent cells and n = 137 for dispersed cells from at least three independent experiment. Cells exceeding by more than 1 SD the average value of confluent cells were considered as active (gray area).

 
To test whether the [Ca2+]i elevations required external Ca2+ or relied on Ca2+ release from intracellular stores, we transiently removed Ca2+ from the bath solution. As shown in Fig. 4BGo, the spontaneous calcium activity of dispersed cells completely disappeared when Ca2+ was removed from the extracellular medium, demonstrating that extracellular calcium is needed for the spontaneous calcium transients. Interestingly, when Ca2+ was subsequently reintroduced, the percentage of activated cells was further increased (Fig. 4Go, B and C). This secondary increase was observed both in dispersed and confluent cells, although the amplitude of the Ca2+ elevations was only slightly above the threshold in confluent cells (Fig. 4Go, A and C). Next, cells were stimulated with 1 µM thapsigargin, an inhibitor of sarcoendoplasmic reticulum Ca2+-ATPases, to assess the content of intracellular Ca2+ stores. Thapsigargin mobilized equivalent amounts of Ca2+ in dispersed and confluent cells (Fig. 4DGo), indicating that the ER Ca2+ content was not altered by cell-cell contact disruption. After exhaustive depletion of ER Ca2+ stores, Ca2+was readmitted to monitor the activity of store-operated Ca2+ entry channels. Robust Ca2+ responses were observed during Ca2+ readmission in both dispersed and confluent cells (Fig. 4Go, E and F). Interestingly, Ca2+-transients were occasionally observed in dispersed cells during the Ca2+ readmission phase, indicating that spontaneous Ca2+ oscillations resembling those observed under basal conditions can occur in cells whose Ca2+ stores had been completely depleted by thapsigargin. Nonetheless, analysis of the amplitude and kinetics of the Ca2+ influx responses did not reveal any significant difference between dispersed and confluent cells at the level of store-operated Ca2+ entry (Fig. 4Go, G and H).


Figure 4
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  		FIG. 4. Effect of Ca2+ removal and readmission on the spontaneous Ca2+ transients. MIN6B1 cells loaded with fura2-AM were subjected to a Ca2+ switch protocol and then exposed to the sarcoendoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin to passively deplete intracellular Ca2+ stores. A and B, Representative Ca2+ recordings of confluent (A) and dispersed cells (B) during Ca2+ removal, Ca2+ readmission, and Ca2+ store depletion. Traces were normalized to baseline (R/Ro). C, Scatter graph of the maximal ratio values recorded during the different phases of the protocol. Each point represents a single cell measurement with n = 58 for confluent cells and n = 61 for dispersed cells from at least three independent experiment. Cells exceeding by more than 1 SD the average value of confluent cells measured before Ca2+ removal were considered as active (gray area). D, Statistical evaluation of the integrated Ca2+ responses [area under the curve (AUC)] evoked by thapsigargin. Bars are mean ± SEM of 58 confluent cells and 61 dispersed cells from three independent experiment. E and F, Representative Ca2+ recordings of confluent (E) and dispersed cells (F). Ca2+ stores were first depleted by the addition of 1 µM TG in Ca2+-free medium, and Ca2+ was added back into the medium to monitor Ca2+ entry. Traces were normalized to baseline (R/Ro). G and H, Statistical evaluation of the integrated Ca2+ responses [area under the curve (G) and slope (H)] evoked by Ca2+ readdition after Ca2+ store depletion. Bars are mean ± SEM of 71 confluent cells and 57 dispersed cells from three independent experiments.

 
Effects of prolonged incubation at low glucose on spontaneous Ca2+ activity and basal insulin release
To test whether we could minimize the spontaneous activity of dispersed cells, they were maintained at 2.8 mM glucose for 6 instead of 2 h before Ca2+ measurements. As shown in Fig. 5Go, the number of activated cells was significantly reduced after prolonged incubation in low glucose (13 vs. 29%, P < 0.05), as was the mean amplitude of the spontaneous Ca2+ transients (1.11 ± 0.03 vs. 1.26 ± 0.04 for 6 h vs. 2 h in low glucose, respectively, P < 0.02). Insulin secretion was measured during three consecutive 90-min incubations in 2.8 mM glucose. Basal release decreased markedly during the 270-min incubation (3.09% of total insulin during the first, 0.68% in the second, and 0.51% in the third 90 min basal incubation, Fig. 5Go). When stimulated at the end of this 270-min period, the dispersed cells responded to glucose with an 11-fold increase in secretion (P < 0.05, Fig. 5Go). When confluent cells were treated in the same manner, basal secretion also decreased (0.8% in the first, 0.4% in the second, and 0.3% in the third 90 min basal incubation), but this was statistically nonsignificant.


Figure 5
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  		FIG. 5. Effect of prolonged incubation at low glucose on spontaneous Ca2+ activity and basal insulin release. A, MIN6B1 cells were loaded with fura2-AM after 2 or 6 h of preincubation in low (2.8 mM) glucose. Scatter graph of the maximal ratio values recorded in confluent and dispersed cells incubated for 2 or 6 h in low glucose (6HLG). Each point represents a single-cell measurement with n = 102 for confluent cells, n = 152 for dispersed cells, and n = 77 for dispersed cells after 6 h in low glucose from at least three independent experiments. Threshold of activation was defined as in Fig. 3Go. B, Insulin secretion from dispersed cells preincubated for 2 h in 2.8 mM glucose, followed by three successive 90-min incubations under basal conditions (2.8 mM glucose, {square}) followed by 90-min at 16.7 mM (stimulated) glucose ({blacksquare}). Results are expressed as in Fig. 1BGo (n = 5 independent experiments).

 
Role of VDCCs in basal insulin release from dispersed cells
When extracellular Ca2+ was removed from the incubation buffer, basal insulin secretion was no longer elevated from dispersed cells (6.2% of basal secretion in CTRL vs. 1.6% in calcium-free condition, P < 0.001, Fig 6AGo). This suggested that Ca2+-uptake is essential for such elevated basal secretion. To verify whether VDCCs are implicated in such Ca2+ entry, nifedipine was used to either inhibit specifically L-type channels (3 µM nifedipine) or more generally all the VDCCs (50 µM nifedipine). As expected and already shown, both concentrations of the drug totally inhibited glucose-induced insulin secretion [11% secretion in control dispersed cells vs. 2% in cells treated with nifedipine 3 µM, 11.7 and 0.6%, respectively, for confluent cells, P < 0.001 (Fig. 6BGo), not shown for nifedipine 50 µM]. However, basal insulin release from dispersed cells was not affected by nifedipine at 3 µM [3.5% in control cells vs. 3% in cells treated with nifedipine, n.s., Fig. 6BGo) or 50 µM (not shown)]. Note that in contrast to earlier experiments (Figs. 1Go and 2Go), in this particular series of experiments, stimulated secretion from dispersed cells was similar to that from confluent cells (Fig. 6Go). However, basal secretion from dispersed cells remained elevated. Whereas the reason for the variability in stimulated secretion from dispersed cells remains to be established, it is possible that more cells had formed small clusters in those series.


Figure 6
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  		FIG. 6. Role of extracellular calcium and VDCCs in basal insulin release from dispersed cells. A, Insulin secretion in response to glucose in presence or absence of extracellular Ca2+ during basal incubation, stimulated secretion in standard KRBH buffer. B, Insulin secretion in response to glucose in presence or absence of 3 µM nifedipine. D, Dispersed; C, confluent; {square}, basal; {blacksquare}, stimulated with 16.7 mM glucose. Data are mean ± SEM of quadruplicate of two independent experiments. *, P < 0.001 vs. control.

 
Cytoskeleton organization and ERK activation in dispersed vs. confluent cells
One of the effects of [Ca2+]i elevations in response to secretagogues is the depolymerization of the actin cytoskeleton to allow the docking of secretory granules to the plasma membrane (33). We therefore investigated cytoskeleton organization in dispersed and confluent cells using fluorescently labeled phalloidin to visualize cortical F-actin by confocal microscopy. Cells were incubated for 2 h at low glucose (2.8 mM) followed or not by 10 min at high glucose (16.7 mM) (Fig. 7AGo). As previously described for MIN6 or MIN6 B1 cells (34, 35), the actin cytoskeleton of confluent cells was well organized under basal conditions (2.8 mM glucose) and partially depolymerized after 10 min stimulation with 16.7 mM glucose (Fig. 7AGo, right panels). By contrast, the actin cytoskeleton of isolated cells was disorganized under basal conditions and did not remodel after stimulation with 16.7 mM glucose (Fig. 7AGo, left panels). However, we observed that dispersed cells began to repolymerize their actin cytoskeleton after a longer period (6 h) in low-glucose medium (Fig. 7BGo). Actin fibers appear visible at this stage in contrast to dispersed cells after only 2 h in low-glucose conditions. Another effect of [Ca2+]i elevation is the rapid phosphorylation of the MAPK member ERK 1/2, which is in turn essential for the early phase of stimulated insulin secretion (35, 36, 37). Believing it possible that the MAPK pathway remained activated for longer in dispersed than in confluent cells after a switch from high to low glucose, possibly accounting in turn for elevated basal secretion, we next investigated how ERK 1/2 phosphorylation evolves when cells are transferred from high- to low-glucose medium. To this end, ERK phosphorylation was measured after 48 h of culture in standard (25 mM) glucose, and the cells were then serum starved for 2 h while maintaining 25 mM glucose to reduce constitutive (serum induced) phosphorylation. Finally, the cells were transferred to low-glucose KRBH. Phosphorylation of ERK1 and ERK2 (Thr-202/Tyr-204 phosphorylation) was analyzed by Western blot. As shown in Fig. 8Go, regardless of their confluency, cells showed a higher phosphorylation of ERK 1/2 in high-glucose medium than after the switch to low glucose. However, and as hypothesized, after switching to the low-glucose condition, ERK activation (phosphorylation) remained high in dispersed cells for up to 240 min, compared with confluent cells, in which ERK phosphorylation was rapidly lost, showing that this pathway remains activated, even without glucose stimulation.


Figure 7
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  		FIG. 7. Differences in actin cytoskeleton organization and remodeling between dispersed and confluent cells. A, Confocal microscopy images showing the differences in the organization and dynamic properties of the actin cytoskeleton in dispersed or confluent cells. In the basal state, cells were fixed after 2 h in 2.8 mM glucose. Dispersed cells show a disorganized actin cytoskeleton, compared with confluent cells, in which actin is well organized in fibers. In the stimulated state, response of cells to 10 min stimulation with 16.7 mM glucose after 2 h in 2.8 mM glucose is shown. The actin cytoskeleton is depolymerized after exposure to glucose in confluent cells, whereas dispersed cells show no remodeling after glucose stimulation. B, Confocal microscopy images showing the differences in the organization of the actin cytoskeleton in dispersed cells after 2 or 6 h in low-glucose conditions. Actin fibers appear visible when dispersed cells have been fixed for 6 h in low glucose but not when the cells have been fixed for 2 h. F-actin was stained with AlexaFluor 546-phalloidin, nucleus with Draq V. Bars, 2 µm.

 

Figure 8
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  		FIG. 8. ERK activation at high or low glucose. After 48 h in culture, dispersed (D) or confluent (C) cells were serum starved for 2 h (25 mM glucose) and then moved to low-glucose medium (KRBH 2.8 mM glucose). Protein extraction was performed either before glucose removal or after 30, 60, 120, or 240 min medium change. ERK1/2 phosphorylation (p-ERK) was determined by Western blotting with specific antiphospho-ERK antibodies, compared with antibodies specific for anti-ERK. High glucose induces phosphorylation of signaling proteins ERK 1/2 in dispersed and confluent cells. ERK 1/2 phosphorylation remained high when dispersed cells were transferred to low-glucose medium in contrast with confluent cells. Data are from a single experiment representative of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Cell-cell contacts within islets are known to be of importance for the normal coordinated regulation of insulin secretion (3, 4, 5, 6). Any perturbation of normal islet architecture impacting on cell-cell communication may thus perturb insulin secretion. One such example could be the fibrosis in the pancreatic islet that plays an important role in diabetes progression and that may alter cellular interactions within islets and notably between β-cells (38). Current attention to cell therapy for diabetes will also have to take cell-cell communication into consideration because the implantation of single β-cells will likely not imitate normal islet function and thus may not allow for normal glucose homeostasis (39). Therefore, it is important to better understand the role of intercellular contacts in the normal function of β-cells and specifically in the regulation of insulin secretion. In this study we used the MIN6 B1 subclone to investigate the mechanism that leads to a more efficient regulation of insulin secretion when cells are in contact. As already described, we show that cell-cell contacts allow the cells to respond better to glucose stimulation and to secrete less insulin under basal conditions, the two essential components of secretion underlying normal glucose homeostasis. We observed that confluent cells were nicely spread, whereas dispersed cells remained round after 48 h in culture, and this may have an impact on insulin secretion. However, dispersed MIN6B1 cells cultured on extracellular matrix [804G matrix (40, 41)] are able to spread, but they still present spontaneous Ca2+ oscillations, elevated basal secretion, and a reduced glucose-stimulated secretion (results not shown). This suggests that loss of contact rather than cell shape is responsible for the insulin secretion defects in dispersed pancreatic β-cells.

Given that cells were always seeded at the same concentration (105 cells per 0.5 ml) and assuming rapid diffusion of any secretory product in these two-dimensional cultures that would preclude ultrashort loop para- or autocrine effects, we conclude that paracrine effects can be neglected and that the above phenotype is mainly due to cell-cell interactions.

The first step was to verify that dispersed cells were not dying and that they did not present problems with insulin synthesis or processing. Quantification of apoptotic cells showed that basal insulin release from dispersed cells is not the result of increased death of isolated cells, compared with confluent cells. There was furthermore no difference in replication between dispersed and confluent cells. We also show that insulin synthesis is not disturbed in isolated cells, in keeping with the hypothesis that fine control of insulin secretion is more due to appropriate release of mature hormone from β-cells than to the acute regulation of insulin biosynthesis (42).

To determine whether cells with intercellular contacts adapt their gene expression program to become more responsive to glucose, we analyzed gene expression using microarray technology. No significant changes in gene expression were found in cells cultured in dense or dispersed conditions for 2 d, suggesting that improved regulation of insulin secretion is a direct consequence of cell-cell interactions, rather than the result of contact-induced changes in gene expression. Our results thus confirm experimentally the hypothesis of a previous study (43). Nonetheless, we cannot exclude that very subtle modulation of expression of genes or gene families could be implicated in the above phenotype. These very small changes might not have been detected due to the limitation of the technique used in this study and possibly due to a limited number of experiments (n = 3). In addition, we chose a time point of 48 h to observe long-term modifications and cannot exclude that changes in gene expression could have occurred at a much earlier time point. In any case, secretion was studied at this same 48-h time point.

Because direct cell-cell contact was shown to be of paramount importance, we decided to down-regulate E-cadherin, one of the major adhesion molecules involved in intercellular contacts between pancreatic β-cells (23). Surprisingly, cells with decreased E-cadherin were apparently still in intimate contact with neighboring cells. These results suggest that either the cells still had enough E-cadherin located on their plasma membranes to be coupled to other cells or that other adhesion molecules may participate in intercellular connections. Down-regulation of E-cadherin had no impact on secretion from dispersed cells, which was to be expected because dispersed cells have few if any cell-cell contacts but led to a significant decrease in glucose-stimulated secretion in confluent cells. Our results suggest that E-cadherin contributes in an important fashion to the proper regulation of insulin secretion and are supported by another report (24). However, we cannot say at this time whether E-cadherin acts in a direct way to promote stimulated secretion or whether the loss of this molecule leads to cosilencing of other proteins that may be implicated in this process. Many papers (44, 45, 46, 47) have shown that regulation of E-cadherin and the gap junction forming proteins connexins is strongly linked. It has also been shown that suppression of Cx36 (the main gap junction molecule in pancreatic β-cells) leads to dysregulation of insulin secretion (48, 49, 50, 51, 52, 53), and indeed, others have suggested that Cx36 and not E-cadherin is essential for insulin secretion (48). We cannot determine from the present study whether E-cadherin is directly implicated in stimulated insulin secretion or whether the decreased secretion after knockdown of this cell adhesion molecule was secondary to modulation of other proteins, possibly including Cx36. A very recent paper has shown that communication between pancreatic β-cells via ephrin-A and EphA plays a major role in insulin secretion (54). Intriguingly, in other cell types, E-cadherin has been shown to specifically regulate Eph receptors and ephrin expression (55). It will thus be most interesting in future studies to investigate the link between all these molecules in β-cells and how this impacts on the regulation of insulin secretion.

Regardless, decrease of E-cadherin expression by 50% had no impact on basal secretion. This may be due to the fact that the cells are still in contact with each other.

[Ca2+]i measurements showed that almost 30% of dispersed cells present spontaneous Ca2+ elevations, whereas only few confluent cells do so. It is well known that once [Ca2+]i has reached a critical threshold insulin secretion is triggered (56). The amplitude of the spontaneous and the glucose-induced Ca2+-elevations observed in dispersed cells was within this range (not shown), suggesting that the spontaneous Ca2+ transients are sufficient to trigger insulin granule release in dispersed cells. Furthermore, several downstream effectors that depend on Ca2+ elevations were activated in dispersed cells in low-glucose conditions.

Based on our results, we propose that a certain percent of dispersed cells present spontaneous Ca2+ activity leading to granule exocytosis at low glucose. Abnormal activity of plasma membrane Ca2+ channels certainly accounts for the spontaneous hyperactivity because these aberrant Ca2+ transients require extracellular Ca2+. Furthermore, Ca2+ transients were still observed in cells whose Ca2+ stores had been depleted by thapsigargin, indicating that the spontaneous Ca2+ oscillations do not require Ca2+ release from stores but Ca2+ influx across membrane channels.

When Ca2+ is removed from extracellular medium, spontaneous Ca2+ elevations as well as basal insulin secretion are abolished. This strongly suggests that these transients are responsible for basal insulin release. It has been shown previously that the membrane potential is different in single cells, compared with cells in islets: dispersed β-cells are more depolarized than cells within islets and therefore most probably more sensitive to depolarization leading to exocytosis (57, 58). Another paper has shown that single β-cells without KATP channels present elevated Ca2+ and basal insulin release (59), suggesting that the rise in basal insulin secretion from isolated cells is due to an inability of quiescent cells to clamp the membrane potential of other β-cells with low stimulation threshold. However, our results using nifedipine seem to exclude the implication of VDCCs in basal insulin secretion. Indeed, even if nifedipine was able to prevent glucose-induced calcium elevation, it was not able to stop spontaneous Ca2+ entry (not shown). Nifedipine totally abolished GSIS but not basal insulin release, suggesting that even if both components of secretion need extracellular Ca2+ entry, the mechanism by which Ca2+ is entering the cell is distinct. This could be of importance to specifically inhibit basal insulin release and efficiently stimulate insulin release.

Our Ca2+ recordings also revealed that dispersed cells exhibiting spontaneous Ca2+ activity failed to mount additional Ca2+ responses when exposed to glucose. In contrast, Ca2+ responses were observed during glucose or KCl stimulation of quiescent cells, but these Ca2+ responses were very heterogeneous, unlike the rapid and homogenous response observed in confluent cells. This suggests that active dispersed cells cannot respond to Ca2+-mobilizing secretagogues, whereas quiescent cells can still be stimulated but less efficiently than confluent cells. In our secretion assay covering a 1-h period, the secretion from quiescent dispersed cells is thus likely masked by the elevated basal secretion from active dispersed cells, whose proportion might well exceed the 28% observed during our shorter (20 min) Ca2+ recordings.

Finally, we show that when cells are maintained for a longer period in the presence of low glucose, the number of dispersed cells with spontaneous Ca2+ activity decreases significantly as does the basal insulin release. This allows the dispersed cells to respond to glucose with an 11-fold stimulation. Even if this response remains weaker than that of confluent cells (an astonishing 39-fold stimulation after 6 h at low glucose), it proves that dispersed cells are able to be stimulated by glucose once basal insulin release has been normalized, showing that they are glucose sensitive and that they keep the capacity to respond to glucose stimulation.

In conclusion, we show here that the enhanced insulin secretory response to glucose in confluent cells is not a cause of any major contact-induced changes in gene expression. Rather, cell-cell interactions appear to be required to decrease spontaneous Ca2+ elevations and to minimize basal insulin secretion, whereas engagement of E-cadherin consequent to cell-cell interaction promotes robust secretion in response to glucose. These findings underline the fundamental importance in normal β-cell function of dual regulation of basal and stimulated secretion by cell-cell contacts. This could be of importance in elucidating the molecular defects underlying the impaired secretion from β-cells in type 2 diabetes that is typified by high basal secretion and poor response to glucose, the hallmark of poorly regulated insulin secretion from dispersed β-cells.


   Acknowledgments
 
We thank Pascale Ribaux for critical reading of the manuscript.


   Footnotes
 
This work was supported by Grant 7-2005-1158 from the Juvenile Diabetes Research Foundation, Grant 310000-11396711 from the Swiss National Science Fund, and an unrestricted educational grant from Novo Nordisk A/S, Gentofte, Denmark.

Disclosure Statement: The authors of this manuscript have nothing to declare.

First Published Online January 24, 2008

1 H.J. and A.T. contributed equally to this work. Back

Abbreviations: [Ca2+]i, Intracellular free calcium concentration; GSIS, glucose-stimulated insulin secretion; hGH, human GH; IBMX, 3-isobutyl-1-methylxanthine; KRBH, Krebs-Ringer bicarbonate HEPES buffer; PMA, phorbol-12-myristate-13-acetate; RNAi, RNA-interference; shRNA, small-hairpin RNA; VDCC, voltage-dependent calcium channel.

Received July 17, 2007.

Accepted for publication January 16, 2008.


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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
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