Article
Reference
Inhibition of calpain blocks pancreatic beta-cell spreading and insulin secretion
PARNAUD, Géraldine, et al.
Abstract
In addition to promoting insulin secretion, an increase in cytosolic Ca(2+) triggered by glucose has been shown to be crucial for spreading of beta-cells attached on extracellular matrix (804G matrix). Calpains are Ca(2+)-dependent cysteine proteases involved in an extended spectrum of cellular responses, including cytoskeletal rearrangements and vesicular trafficking. The present work aimed to assess whether calpain is also implicated in the process of Ca(2+)-induced insulin secretion and spreading of rat pancreatic beta-cells. The results indicate calpain dependency of beta-cell spreading on 804G matrix. Indeed, treatment with three distinct calpain inhibitors (N-Ac-Leu-Leu-norleucinal, calpeptin, and ethyl(+)-(2S,3S)-3-[(S)-3-methyl-1-(3-methylbutylcarbamoyl)butyl-carbamoyl]-2-ox-iranecarbo xylate) inhibited cell spreading induced by glucose and KCl, whereas cell attachment was not significantly modified. Calpain inhibitors also suppressed glucose- and KCl-stimulated insulin secretion without affecting insulin synthesis. Washing the inhibitor out of the cell culture restored spreading on 804G matrix and insulin [...]
PARNAUD, Géraldine, et al . Inhibition of calpain blocks pancreatic beta-cell spreading and insulin secretion. American Journal of Physiology. Endocrinology and Metabolism , 2005, vol. 289, no. 2, p. E313-21
DOI : 10.1152/ajpendo.00006.2005 PMID : 15784646
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Inhibition of calpain blocks pancreatic  -cell spreading and insulin secretion
Ge´raldine Parnaud,1Eva Hammar,1Dominique G. Rouiller,1and Domenico Bosco2
1Department of Genetic Medicine and Development, University Medical Center; and2Cell Isolation and Transplantation Center, Division of Surgical Research, Department of Surgery, University Hospital, Geneva, Switzerland
Submitted 7 January 2005; accepted in final form 14 March 2005
Parnaud, Ge´raldine, Eva Hammar, Dominique G. Rouiller, and Domenico Bosco. Inhibition of calpain blocks pancreatic
-cell spreading and insulin secretion. Am J Physiol Endocrinol Metab 289: E313–E321, 2005. First published March 22, 2005;
doi:10.1152/ajpendo.00006.2005.—In addition to promoting insulin secretion, an increase in cytosolic Ca2⫹triggered by glucose has been shown to be crucial for spreading of-cells attached on extracellular matrix (804G matrix). Calpains are Ca2⫹-dependent cysteine pro- teases involved in an extended spectrum of cellular responses, includ- ing cytoskeletal rearrangements and vesicular trafficking. The present work aimed to assess whether calpain is also implicated in the process of Ca2⫹-induced insulin secretion and spreading of rat pancreatic
-cells. The results indicate calpain dependency of-cell spreading on 804G matrix. Indeed, treatment with three distinct calpain inhibi- tors (N-Ac-Leu-Leu-norleucinal, calpeptin, and ethyl(⫹)-(2S,3S)-3- [(S)-3-methyl-1-(3-methylbutylcarbamoyl)butyl-carbamoyl]-2-ox- iranecarboxylate) inhibited cell spreading induced by glucose and KCl, whereas cell attachment was not significantly modified. Calpain inhibitors also suppressed glucose- and KCl-stimulated insulin secre- tion without affecting insulin synthesis. Washing the inhibitor out of the cell culture restored spreading on 804G matrix and insulin secre- tory response after 24 h. In addition, incubation with calpeptin did not affect insulin secretory response to mastoparan that acts on exocytosis downstream of intracellular calcium [Ca2⫹]i. Finally, calpeptin was shown to affect the [Ca2⫹]iresponse to glucose but not to KCl. In summary, the results show that inhibition of calpain blocks spreading and insulin secretion of primary pancreatic -cells. It is therefore suggested that calpain could be a mediator of Ca2⫹-induced-insulin secretion and-cell spreading.
extracellular matrix; intracellular calcium
CALPAINS ARE CYTOSOLIC NEUTRALCa2⫹-dependent cysteine pro- teases that catalyze the endoproteolytic cleavage of specific substrates and thereby regulate pathways that affect intracel- lular signaling (25, 28). Fourteen members of the calpain family have been identified, some of which are tissue specific whereas others are ubiquitously expressed (12). The most widely distributed calpains consist of two major isoforms, calpain I, or -calpain, and calpain II, or m-calpain, which reflect their sensitivity to activation by micromolar and milli- molar calcium concentrations, respectively. Calpains are ex- pressed in a variety of mammalian cells, including -cells of pancreatic islets (19).
Calpain activity has been shown to be crucial in a diverse spectrum of cellular responses. By making selective limited proteolytic cleavages, calpains modulate the activity of en- zymes and induce specific cytoskeletal rearrangements, ac- counting for their roles in cell spreading and motility, vesicular
also expressed in rat pancreatic acinar cells and may participate in stimulus-secretion coupling of amylase (42).
Various forms of calpain have also been implicated in pathological processes, such as neurodegenerative disorders, Alzheimer’s disease, and muscular dystrophy (16, 26, 31, 40).
Variation in CAPN10, the gene encoding the ubiquitously expressed cysteine protease calpain-10, has been associated with type 2 diabetes in Mexican Americans and in two north- ern-European populations, from Finland and Germany (15).
The extracellular matrix (ECM) plays a critical role in modulating the morphology, growth, migration, and differen- tiation of cells. The response of cells to matrix-adhesive proteins appears to be mediated primarily through the integrin family of adhesion receptors (24). The ␣3- and ␣6-integrins have been shown to be expressed in rat pancreatic-cells (3, 18) and are known to be the principal receptors for the ECM protein laminin. Furthermore, rat islet -cells spread in re- sponse to glucose when attached on the matrix produced by a rat bladder carcinoma cell line (804G), known to be rich in laminin 5. In a mixed population of cells, it has been previously observed that spread cells secrete more insulin acutely in response to glucose compared with cells that remain rounded (3). Glucose-induced spreading of -cells on 804G matrix requires an increase in intracellular calcium concentration ([Ca2⫹]i) and is not a consequence of exocytotic processes that follow elevation of [Ca2⫹]i(2).
It is well known that Ca2⫹ plays a crucial role in insulin secretion from -cells. A significant influx of Ca2⫹ occurs during the process of glucose-mediated insulin secretion. A wide variety of cytoplasmic enzymes, mostly kinases, are known to be activated or modulated by an elevation of [Ca2⫹]i
and have been also identified in -cells (17). These include calcium/calmodulin- and calcium/phospholipid-dependent ki- nases that have been shown to be involved in the initiation and maintenance of nutrient-stimulated insulin secretion. Whether calpain may also be implicated in the process of Ca2⫹-induced insulin secretion is not yet understood. In recent studies, calpain has been implicated in the process of exocytosis in the insulin-secreting cell line INS-1 or in mouse islet (23, 27, 38, 43). The purpose of this study was to assess the implication of a calpain pathway in spreading and secretion of purified insu- lin-secreting-cells of the rat pancreas.
MATERIALS AND METHODS
804G matrix preparation.Cells from a rat bladder carcinoma cell line (804G) were grown in DMEM containing 10% FCS and 5.6 mM glucose (32). At confluence, cells were washed and maintained for a First published March 22, 2005; doi:10.1152/ajpendo.00006.2005.
medium (referred to hereafter as 804G matrix) was collected, centri- fuged at 180 gfor 5 min to remove any detached cells and debris, filtered through a 0.22-m Millipore filter, and frozen at⫺20°C for later use.
Coating of plastic with 804G matrix and poly-L-lysine.Aliquots (60
l) of 804G matrix or 100g/ml poly-L-lysine (PLL) were layered at the center of 35-mm adherent-culture Petri dishes. Dishes were kept in a damp box at 37°C for 18 –20 h before being washed three times with sterile H2O and air dried. PLL-coated Petri dishes were used as a control.
Islet isolation. Islets of Langerhans were isolated by collagenase digestion of pancreases from male Wistar rats (weighing 180 –200 g, Janvier, Le Genest-St-Isle, France) followed by Ficoll purification using a modification of the method of Sutton et al. (39). For cell preparation, the isolated islets were trypsinized and -cells purified using a FACStar-Plus cell sorter (Becton-Dickinson) as previously described (35, 41).
Cell culture.Sorted -cells were washed twice with sterile Dul- becco’s minimum essential medium (DMEM) (GIBCO, Invitrogen, Basel, Switzerland) containing 10% heat-inactivated FCS, 11.2 mM glucose, supplemented with 1 mM sodium pyruvate, 110 U/ml peni- cillin, 110g/ml streptomycin, and 50g/ml gentamicin (referred to hereafter as control medium), followed by centrifugation for 5 min at 180 g. Aliquots of 105 cells were seeded in nonadherent 60-mm- diameter Petri dishes containing 3 ml of control medium. Cells were then incubated for 20 h at 37°C to allow full recovery of any cell surface molecules that might have been lost or damaged during islet isolation or cell purification.-Cells were then centrifuged for 5 min at 180gand resuspended at a density of 8⫻105cells/ml in control DMEM supplemented or not with a calpain inhibitor: ALLN (calpain inhibitor I, N-Ac-Leu-Leu-norleucinal; Calbiochem, Juro, Switzer- land), calpeptin (Calbiochem), or EST (ethyl(⫹)-(2S,3S)-3-[(S)-3- methyl-1-(3-methylbutylcarbamoyl)butylcarbamoyl]-2-oxirane carboxy- late, Calbiochem). Aliquots (50l) of this suspension were plated as droplets at the center of Petri dishes coated with 804G matrix or PLL and were incubated at 37°C. After 24 h, cells were analyzed for attachment and spreading under an inverted microscope. The calpain
inhibitors were dissolved in DMSO, which was also added to control cultured cells at the same concentration. In addition, medium (⬃50
l) was collected and centrifuged to remove any detached cells and debris and stored at⫺20°C for subsequent insulin measurement.
Actin staining.Actin was detected by staining with Alexa Fluor 546 phalloidin (Molecular Probes). After 24 h of attachment, the cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min, permeabilized with 0.5% Triton X-100 in PBS for 4 min, and then stained with Alexa Fluor phalloidin. Images were collected using a Zeiss confocal laser-scanning microscope.
Insulin secretion assay and insulin content.After 24 h of incuba- tion, cells were washed three times with a 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, 0.1% BSA) supplemented with 2.8 mM glucose and prein- cubated with this same buffer for 1 h at 37°C. Cells were then incubated for 1 h at 37°C with KRBH containing 2.8 mM glucose and successively for 1 h at 37°C with KRBH containing 16.7 mM glucose or with 2.8 mM glucose supplemented with 10M mastoparan or 20 mM KCl. The incubation buffer was recovered and insulin extracted from cells with acid-ethanol. The amounts of insulin in the incubation buffer and cellular extracts were measured by classical radioimmu- noassay using the charcoal separation technique. Rat insulin was used as the standard with a guinea-pig anti-porcine insulin antibody and
125I-labeled insulin as the tracer. Insulin content is expressed as the sum of insulin extracted at the end of the stimulation test and insulin secreted during the first and second incubation periods. Data are expressed as means⫾SE.
Quantification of insulin mRNA by real-time PCR.Total RNA of
-cells in the culture dish was isolated using the QIAshredder and RNeasy Mini Kit (Qiagen), according to the instructions of the manufacturer, and stored at⫺80°C. The RNA quality was verified by agarose gel electrophoresis. First-strand cDNA was synthesized by oligo(dT) priming of 1g of total RNA using the Superscript II RT kit (Invitrogen) according to the manufacturer’s protocol. PCR am- plification was performed with the iCycler-iQ system (Bio-Rad).
Primers were designed according to the Primer Expresss software.
Fig. 1. Effect of calpeptin on pancreatic
-cell spreading. -Cells were plated on poly-L-lysine (PLL; A–C) or 804G matrix (D–I) and incubated for 24 h under basal condition (2.8 mM glucose and 5 mM KCl;
AandD), 11.2 mM glucose and 5 mM KCl (BandE), or 2.8 mM glucose and 25 mM KCl (CandF). Addition of 35g/ml cal- peptin inhibited spreading induced both by glucose (H) or KCl (I) and had no effect on morphology of cells incubated under basal condition (G). Bar, 10m.
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qPCR core Kit SYBR Green I (Eurogentech) supplemented with fluorescein (Bio-Rad) was used in the PCR reaction to allow for quantitative detection of the PCR product. Rat insulin primers used were 5⬘-CTGCCCAGGCTTTTGTCAAA-3⬘and 5⬘-TCCACTTCAC- GACGGGACTT-3⬘as sense and antisense primers, respectively. The temperature profile of the reaction was 95°C for 10 min, 45 cycles of denaturation at 95°C for 15 s, and annealing/extension at 60°C for 1 min. An internal housekeeping gene control, ribosomal RNA L3, was used to normalize differences in RNA isolation, RNA degradation, and the efficiencies of the reverse transcription. All results were expressed as the ratios of the copy number of insulin to that of the housekeeping gene L3.
Measurement of [Ca2⫹]i. Cells attached on glass coverslips for 24 h were loaded in KRBH containing 2.8 mM glucose for 30 min at room temperature in the presence of 1.5 M fura 2-acetoxymethyl ester (Molecular Probes, Eugene, OR). The coverslips were washed,
cence emission at 510 nm was captured every 5 s using a cooled, back-illuminated, 16-bit, charge-coupled device, frame transfer cam- era (Princeton, Roper Scientific, Trenton, NJ) controlled by Meta- morph/Metafluor 4.1.2 software (Universal Imaging, West Chester, PA). By use of established procedures, changes in the emission intensity of fura 2, expressed as a ratio of dual excitation, were used as indicators of changes in [Ca2⫹]i. Three independent experiments were performed on each group, with 40 cells examined for each experiment.
Calpain activity assay.Intracellular calpain activity was measured using a commercial kit (Calpain Activity Assay Kit; Oncogene, Juro, Switzerland) according to the manufacturer’s instructions. This kit is a fluorometric assay using a calpain substrate, the fluorogenic peptide succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY- AMC).
Calpain activity of individual-cells was determined by incubating the cells with the calpain substrate BOC-Leu-Met-CMAC. This sub- strate readily permeates biological membranes and, once inside the cell, conjugates with glutathione to form BOC-Leu-Met-CMAC-SG (34). Calpain cleaves the conjugate between methionine and CMAC- SG, resulting in the fluorescent product CMAC-SG, which has an emission maximum at 410 nm when excited at 360 nm. Cells were plated on coated Petri dishes and incubated for 24 h in the presence or absence of calpeptin. The cells were then incubated for 20 min in the presence of 50 M BOC-Leu-Met-CMAC (Molecular Probes). The
Fig. 2. Dose-dependent and reversibility effect of calpeptin on cell spreading.
-Cells were plated on 804G matrix and incubated for 24 h in medium containing 11.2 mM glucose (A). Calpeptin at 20g/ml (B), 35g/ml (C), and 50 g/ml (D) dose-dependently inhibited spreading induced by glucose.
-Cells were incubated for 48 h in medium containing 11.2 mM glucose in either the absence (E) or presence (F) of 35g/ml calpeptin.G:-cells were incubated in medium containing 11.2 mM glucose for 24 h in the presence of 35 g/ml calpeptin and then for 24 h in the absence of calpeptin. Bar, 100m.
cells were wet mounted with a coverslip and observed for CMAC fluorescence using a Zeiss fluorescent microscope (model Axiophot).
RESULTS
-Cell spreading on 804G is inhibited by calpain inhibitors.
Calpeptin, a calpain inhibitor, was used to define the role of calpain in the adhesion and spreading of pancreatic-cells on the 804G matrix.-Cells rapidly spread out (2–3 h), develop- ing lamellipodia and continuously expressed this phenotype when attached on 804G matrix and incubated in the presence of 11.2 mM glucose (Figs. 1Eand 2A) or 25 mM KCl (Fig. 1F), conditions known to increase [Ca2⫹]i. Under these conditions, addition of calpeptin largely inhibited cell spreading but did not affect cell attachment (Figs. 1,HandI, and 2,B–D). This effect was observed after 2–3 h (not shown), 24 h (Figs. 1,G–I, and 2, B–D), or 48 h (Fig. 2F) of treatment and was dose
dependent (Fig. 2, B–D). -Cell spreading was recovered within 24 h after the calpain inhibitor was removed from the medium, indicating that this inhibition of spreading is revers- ible (Fig. 2G).
Spreading did not occur when-cells were cultured on the 804G matrix at low glucose (2.8 mM; Fig. 1D) or when attached under any condition on PLL (Fig. 1, A–C). Under these conditions, treatment with calpeptin had no effect on
-cell adhesion or morphology (Fig. 1G). The organization of actin filaments in round and spread cells was examined by staining with fluorescent phalloidin. Spread cells contained many stress fibers and microfilaments that were concentrated in lamellipodia (Fig. 3,CandD). In the presence of calpeptin, the organization of actin filaments was modified so that stress fibers were absent (Fig. 3,EandF). Round-cells cultured on the 804G matrix at low glucose show the same actin organi- zation as cells treated with calpeptin (Fig. 3,A andB).
Similar results were observed when the calpain inhibitors ALLN or EST were used instead of calpeptin (data not shown).
To establish the efficacy of calpeptin as an inhibitor of intracellular calpain in -cells, we evaluated its ability to inhibit calpain cleavage of the calpain substrate Suc-LLVY- AMC. Under control conditions, calpain activity was detected in-cells; after addition of calpeptin, a significant decrease of calpain activity was observed (41 ⫾ 12.7% of control in calpeptin-treated-cells, P⬍ 0.01). The intracellular calpain activity in individual -cell was determined by using the calpain substrate BOC-Leu-Met-CMAC. Heterogeneous label- ing with strongly and weakly fluorescent cells was observed (Fig. 4A). However, in -cells treated with calpeptin, only
Fig. 4. Effect of calpeptin on calpain activity in-cells. Calpain activity was determined in cells with the substrate BOC-Leu-Met-CMAC (50M). Cells were incubated for 24 h in the absence (A) or presence (B) of 35 g/ml calpeptin. Cells were then incubated for 20 min with 50M BOC-Leu-Met- CMAC and observed for CMAC fluorescence using a fluorescent microscope.
This figure is representative of 3 different experiments.
Fig. 5. Effect of calpain inhibitors on insulin secretory response to glucose.A, C,andD:
-cells were attached on either PLL or 804G matrix (Mx) and incubated for 24 h with various concentrations of calpeptin (CALP;
A) ethyl(⫹)-(2S,3S)-3-[(S)-3-methyl-1-(3- methylbutylcarbamoyl)butyl-carbamoyl]-2- oxiranecarboxylate (EST; C), and N-Ac- Leu-Leu-norleucinal (ALLN; D); insulin secretion in response to 2.8 mM glucose (open bars) and 16.7 mM glucose (filled bars) was measured. B:-cells were incu- bated for 24 h with 35g/ml calpeptin and successively for 24 h in the absence of cal- peptin, and then insulin secretion in response to 1 h with 2.8 mM glucose (open bars) and 16.7 mM glucose (filled bars) was measured.
Results are means⫾SE of 3–5 experiments in each group. *P⬍0.05 vs. cells incubated in the absence of calpain inhibitors and plated on PLL.
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occasional, weak labeling could be observed (Fig. 4B). These results demonstrate the efficacy of calpeptin to inhibit calpain in intact-cells.
Insulin secretion is inhibited by chronic exposure of-cells to calpain inhibitors. To study the role of calpain on-cell function, -cells were plated on PLL or 804G matrix and preincubated for 24 h with calpeptin, and short-term insulin secretion was then measured after 1-h of incubation at 2.8 mM glucose and 1 h at 16.7 mM glucose. Compared with control, calpeptin dose-dependently inhibited the insulin secretory re- sponse to 16.7 mM glucose of-cells attached on either 804G matrix or PLL (Fig. 5A). The inhibitory effect of calpeptin on insulin secretion thus seems to be independent of the substrate on which the cells are plated. Significant effects on insulin secretion were also observed when ALNN or EST were used (Fig. 5,CandD).
As shown in Table 1, insulin content was significantly increased in-cells treated with 35g/ml calpeptin compared with control. However, there were no significant differences in insulin mRNA levels. Insulin secreted in the medium during the 24-h incubation was significantly reduced in calpeptin- treated cells, suggesting a blockage of the exocytosis step.
To test whether the inhibitory effect of calpeptin on insulin secretion could be reversed,-cells that had been exposed to 35g/ml calpeptin for 24 h were cultured for another 24 h in control medium. Removal of the inhibitor from the cells for 24 h restored insulin secretory responses to levels not different from control (Fig. 5B), indicating that calpain inhibitors do not cause-cell death or irreversible nonspecific toxic effects. By contrast, exposing cells to calpeptin continuously for 48 h did inhibit the insulin secretory response to 16.7 mM glucose (P⫽ 0.038).
To further determine the specificity of calpeptin on Ca2⫹- induced insulin secretion,-cells were stimulated with 25 mM KCl or 10 M mastoparan instead of 16.7 mM glucose. As expected, depolarization of the -cells by KCl stimulated insulin secretion, and the effect was higher in-cells attached on the 804G matrix compared with PLL. Calpeptin treatment inhibited the effect of KCl on insulin secretion in-cells plated on either PLL or 804G matrix (Fig. 6B). Mastoparan, known to activate exocytosis downstream of [Ca2⫹]ielevation, strongly increased insulin secretion in-cells attached on PLL and to a lesser extent in-cells attached on the 804G matrix (Fig. 6C).
Interestingly, calpeptin treatment did not affect mastoparan- induced insulin secretion in-cells attached on PLL or 804G matrix (Fig. 6C). These results suggest that calpain acts up- stream of the exocytotic process but downstream of the rise in intracellular calcium.
Fig. 6. Effect of calpeptin on the insulin secretory response to glucose, KCl, and mastoparan.-Cells were attached on either PLL or 804G matrix (Mx) and incubated for 24 h with 35g/ml calpeptin; insulin secretion in response to 1 h with 16.7 mM glucose (A), 25 mM KCl (B), or 10M mastoparan (C) was then measured. Results are means⫾SE of 4 –7 independent sets of experi- ments.
Table 1. Effect of calpeptin on insulin mRNA expression, insulin contents, and insulin secretion after 24 h of culture
Insulin mRNA, insulin/
L3 mRNA ratio
Insulin Content, ng/20,000 cells
Secreted Insulin Content, ng PLL 1,863.17⫾177.24 439.24⫾18.29 1,408.37⫾190.79 PLL⫹CALP 1,985.43⫾233.16 1,036.13⫾35.36† 76.01⫾22.61*
secretory response to 16.7 mM glucose or 25 mM KCl (Fig. 7), whereas no significant effect was observed on basal insulin secretion (2.8 mM glucose). The inhibition of insulin secretion was observed on cells plated on both the 804G matrix and PLL.
Insulin content was similar in control and in calpeptin-treated cells (data not shown).
[Ca2⫹]i is not directly affected by calpeptin in -cells. To exclude the idea that the deficient insulin secretion and the inhibition of spreading in calpeptin-treated cells are associated with an alteration in intracellular [Ca2⫹]i signaling, we mea- sured [Ca2⫹]i responses to KCl and glucose in absence or presence of calpeptin. -Cells plated on the 804G matrix showed a rise in [Ca2⫹]iin response to KCl similar to that of
-cells plated on PLL (Fig. 8,AandB). The magnitude of the [Ca2⫹]iresponse to KCl was not reduced in calpeptin-treated cells plated on 804G matrix or on PLL. Similarly, exposure to
calpeptin did not reduce the number of cells that showed a rise in [Ca2⫹]iin response to KCl (Table 2). When stimulated with glucose, calpeptin-treated cells still showed an increase of [Ca2⫹]i, even if the magnitude of [Ca2⫹]i response (Fig. 8,C andD) and the number of cells showing a rise in [Ca2⫹]i(Table 2) were lower compared with control. This effect of calpeptin on glucose-induced [Ca2⫹]i elevation suggests that calpeptin may be involved in some pathway coupling glucose to [Ca2⫹]i
elevation.
DISCUSSION
In the present study, we investigated the role of calpain in the process of Ca2⫹-induced insulin secretion and spreading of primary rat -cells. Our data provide evidence that calpain is necessary for glucose- and KCl-induced insulin secretion and
Fig. 7. Effect of short time exposure of
-cells to calpeptin on insulin secretory re- sponse to glucose and KCl. -Cells were allowed to attach on PLL or 804G Mx for 24 h under standard conditions without cal- peptin. Then insulin secretion in response to 1 h at 16.7 mM glucose (A) or 25 mM KCl (B) was tested in the absence or presence of 35 g/ml calpeptin (CALP). Results are means⫾SE of 4 experiments in each group.
Fig. 8. Effect of calpain inhibition on [Ca2⫹]iresponse to KCl and glucose. Cells were cultured for 24 h on glass coverslips coated with PLL (BandD) or 804G Mx (A andC) and were loaded (30 min) with fura 2 under basal conditions. [Ca2⫹]iis expressed as ratio 340/380 nm for fluorescence emis- sion measured every 5 s before and after stimulation with 25 mM KCl (AandB) or 16.7 mM glucose (CandD) (arrow). Values are means of 100 cells analyzed in 3 inde- pendent experiments.
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spreading of rat -cells. Indeed, three distinct, synthetic cell- penetrating calpain inhibitors, calpeptin, ALLN, and EST, block both glucose- and KCl-induced insulin secretion and cell spreading in a dose-dependent manner. The calpain peptidyl inhibitors used in these studies, EST, ALLN, and calpeptin, were chosen on the basis of their specificities. All of these inhibitors inhibit only cysteine proteases. Calpeptin is a hydro- phobic cell-permeable peptidyl aldehyde, containing a ben- zyloxycarbonyl residue at the NH2terminus. The mechanism of action probably involves a covalent interaction between the active-site cysteine of calpain and an electrophilic center of the inhibitor (31). A number of our results point strongly to an inhibition of calpain as the mechanism underlying the observed effects. First, different calpain inhibitors (calpeptin, ALLN, and EST) that work by different mechanisms have similar effects on Ca2⫹-induced spreading and insulin secretion. Sec- ond, hydrolysis of the calpain-specific substrate Suc-LLVY- AMC was inhibited in calpeptin-treated cells. The possibility that nonspecific toxic effects were involved in the impairment of insulin secretion in-cells exposed to calpain inhibitors for 24 h was also considered. We believe that this possibility can be excluded for the following reasons. First, removal of the calpain inhibitors from the medium resulted in the recovery of the insulin secretory response to glucose, making it unlikely that decreased-cell viability is responsible for the defects in insulin secretion. Second, inhibitor-treated cells had normal Ca2⫹responses to the direct depolarization induced by 25 mM KCl. Whether inhibitors used in this study act exclusively on calpain activity was not studied. For example, calpeptin has been shown to inhibit cathepsin B (36). Consequently, we cannot exclude the possibility that other proteases may also contribute in part to the process.
Cleavage of cytoskeletal/membrane attachments is the most thoroughly documented function of the calpains. Work on platelets has shown that the cysteine protease calpain is one of the signaling molecules activated following integrin-ligand interactions (37), suggesting that it may modulate intracellular events required for effective cell adhesion and spreading. The present work suggests that calpain is involved in the spreading of -cells on the 804G matrix. A role of calpain in cell spreading has been described previously in other cell types.
Studies in which calpain was inhibited with membrane-perme- able inhibitors or by an overexpression of the endogenous
hesion kinase (4). The1-integrin cytoplasmic domain is also a target of calpain (29). Interestingly, this integrin is expressed by -cells and has been shown to be involved in -cell spreading and insulin secretion (3). Furthermore, it has been demonstrated that calpain associates with1-integrin-contain- ing membrane lipid rafts (14). This would represent an ideal target for calpain to modulate cell morphology subsequent to signaling from the extracellular matrix.
Calpains also play a role in signal transduction pathways by coupling calcium influxes to important structural alterations of membrane proteins associated with fusion and secretory events. Thus calpain activation has been implicated in protein secretion of neuroblastoma cells (5), rat pancreatic acinar cells (42), and alveolar epithelial cells (44). As in other studies (23, 27, 38, 43), our results show an involvement of calpain in insulin secretion. Some works (23, 43), including ours, show that chronic exposure to calpain inhibitors decreases insulin secretion. However, the effect of an acute exposure to calpain inhibitors on insulin secretion is still controversial. Some studies demonstrated enhancement of glucose-induced insulin secretion in islets exposed for 4 h to calpain inhibitors (38, 43), whereas other studies (27), including ours, showed that calpain inhibitors decreased insulin secretion after an acute treatment.
The reason for these differences is still unknown.
Calpain inhibitors suppressed glucose- and KCl-stimulated insulin secretion without affecting mastoparan-induced insulin secretion. Mastoparan, a tetradecapeptide from wasp venom, is thought to stimulate insulin release by activating small G proteins of the Rho family, such as Cdc42 and Rac (1, 20). Our observation that calpeptin did not affect mastoparan-induced insulin secretion is consistent with the hypothesis that calpain acts at a point upstream of Rho family members (21, 22).
Calpeptin did not affect the KCl-induced [Ca2⫹]iincrease of
-cells; however, calpeptin did decrease the Ca2⫹response of
-cells stimulated with glucose. Zhou et al. (43) demonstrated that calpain inhibition induced a decrease in glucose metabo- lism that may account for the calpeptin effect on the glucose- increased [Ca2⫹]i. Many processes in the signaling pathways of insulin secretion could be affected by calpain inhibition.
Indeed, because the Ca2⫹ response to KCl was normal in calpeptin-treated cells, the effect on secretion of calpeptin likely occurs in signaling events following the rise of [Ca2⫹]i, most likely at the exocytotic process. In short-term studies, the ability of the calpain inhibitors to block only stimulated insulin secretion may result from a specific role for calpain in initial events enabling the priming of undocked rather than predocked granule stores. This hypothesis is in accord with the fact that insulin content was significantly increased in calpeptin-treated cells for 24 h and insulin secreted in the medium during attachment was significantly reduced in calpeptin-treated cells.
The site of calpain action important for -cell spreading is unknown. In our study, calpain did not seem to be implicated in a direct connection between spreading and insulin secretion.
Indeed insulin secretion was also inhibited by calpeptin in nonspread cells. This result is in agreement with a recent study demonstrating that calpain-10 is a regulator of exocytosis in Table 2. Effect of calpeptin on percentage of-cells
showing a [Ca2⫹]iresponse to glucose and KCl
%Cells Responding to
Glucose KCl
PLL 96.4⫾5.0 100⫾0
PLL⫹CALP 56.5⫾25.1* 99⫾2
Mx 100⫾0 100⫾0
Mx⫹CALP 69.1⫾13.1* 98⫾2.7
Data are averages⫾SE of 3 independent experiments. [Ca2⫹]i, intracellular Ca2⫹concentration. *P⬍0.01 vs. its respective control.
spreading: calpain activity was implicated in Ca2⫹-associated secretion events occurring during the first 30 s of platelet activation by the thrombin receptor agonist peptide, and cal- pain activity also regulated later platelet activation events involving integrin function as well as actin remodeling (6). A rise in [Ca2⫹]i may not be a consequence of spreading in response to glucose, because a rise in [Ca2⫹]i in response to glucose has been observed in-cells in suspension (13), and it has been routinely observed in -cells attached to plastic or glass, where they do not spread. A rise in [Ca2⫹]i was also observed in calpeptin-treated-cells, suggesting that calpains may play an important, early role in insulin secretion and regulates later events associated with spreading. In addition, insulin secretion and spreading of-cells, responses inhibited by calpeptin, could each be regulated by different types of calpains.
In conclusion, our results suggest a role of calpains in the regulation of spreading and insulin secretion of primary pan- creatic -cells. The effect of calpains on insulin secretion seems independent of-cell spreading, since insulin secretion of nonspread -cells attached on PLL was also inhibited by calpeptin. Because many steps could be modulated by calpains, further experiments are needed to clarify the molecular and physiological mechanisms associating calpains with insulin secretion and-cell spreading.
ACKNOWLEDGMENTS
We thank Caroline Raveraud and Nicolas Wu¨thrich for technical assistance and Profs. Philippe Halban and Barbara Yermen for critical reading of the manuscript.
GRANTS
This work was supported by Swiss National Science Foundation Grant 3200B0-100656/1 (D. G. Rouiller) and Juvenile Diabetes Research Foundation Grant 1-2000-692 (D. G. Rouiller).
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