Increased Intracellular Calcium Is Required for Spreading of …Increased Intracellular Calcium Is...

Increased Intracellular Calcium Is Required forSpreading of Rat Islet b-Cells on Extracellular MatrixDomenico Bosco,

1Carmen Gonelle-Gispert,

1Claes B. Wollheim,

2Philippe A. Halban,

1

and Dominique G. Rouiller1

Rat islet b-cells spread in response to glucose whenattached on the matrix produced by a rat bladder carci-noma cell line (804G). Furthermore, in a mixed popula-tion of cells, it has been observed previously that spreadcells secrete more insulin acutely in response to glu-cose, compared with cells that remain rounded. Theseresults suggest bi-directional signaling between the is-let b-cell and the extracellular matrix. In the presentstudy, the role of increased intracellular free Ca21

concentration [Ca21]i as an intracellular step linkingglucose stimulation and b-cell spreading (inside-outsignaling) was investigated. Purified rat b-cells wereattached to this matrix and incubated under variousconditions known to affect [Ca21]i. The effect of glucoseon b-cell spreading was mimicked by 25 mmol/l KCl(which induces calcium influx) and inhibited by diazox-ide (which impairs depolarization and calcium entry)and by the L-type Ca21 channel blocker SR-7037. When a24-h incubation at 16.7 glucose was followed by 24 h at2.8 mmol/l, b-cells that had first spread regained a roundphenotype. In the presence of thapsigargin, spreadingprogressed throughout the experiment, suggesting thatcapture of calcium by the endoplasmic reticulum isinvolved in the reversibility of spreading previouslyinduced by glucose. Spreading was still observed indegranulated b-cells and in botulinum neurotoxin E–expressing b-cells when exocytosis was prevented. Insummary, the results indicate that increased [Ca21]i isrequired for the glucose-induced spreading of b-cells on804G matrix and that it is not a consequence of exo-cytotic processes that follow elevation of [Ca21]i.Diabetes 50:1039–1046, 2001

Extracellular matrix (ECM) surrounds pancreaticislets and forms the basal lamina, which sepa-rates endocrine cells from capillaries. Althoughthe molecular components of these basal lami-

nae remain to be fully investigated in islets, there is

evidence indicating that some of the molecules generallyfound in epithelial basal lamina play a role in the growthand differentiation of b-cells. Fetal pancreatic epitheliacultured in Matrigel or collagen I gel differentiated intoendocrine cells and form islet structures (1); neonatal ratislet cells embedded in collagen I formed well-organizedislet-like organoids (2); and adult human b-cells or asso-ciated islet cells proliferated when attached on 804Gmatrix produced by a rat bladder carcinoma cell line (3,4).More recently, it has been shown that laminin I specificallypromotes differentiation of mouse b-cells from precursorcells in vitro. This finding is of physiological relevancebecause laminin I is expressed in basement membranes ofthe mouse pancreas from ;15 days of intrauterine life (5).

The importance of cell-matrix interactions for an opti-mal regulated insulin secretion is supported by a numberof studies on b-cells cultured on various crude matrixes ortheir purified extracts (6–14). In a recent study, we ob-served that b-cells attached to a matrix produced by a ratbladder carcinoma cell line 804G (hereafter referred to as804G matrix) secreted more insulin than control cells. Wealso noticed that on 804G matrix, cells that had spreadexpressed higher amounts of a6b1 integrins and secretedmore insulin than cells that had remained round (15). Theimportance of cell-matrix interactions for regulated insulinsecretion was further demonstrated by the fact that secre-tagogues, including glucose, increased the number ofspread cells (15).

The mechanism leading stimulated b-cell to spread on804G matrix is totally unknown. In the present study, weraise the hypothesis that some of the intracellular path-ways governing stimulation-secretion coupling of b-cellsmight be shared for coupling stimulation and spreading.Intracellular free Ca21 concentration [Ca21]i plays a cen-tral role in the consensus model for stimulus-secretioncoupling, which may be summarized as follows: in re-sponse to glucose, b-cell metabolism is stimulated, result-ing in increased levels of ATP produced by the mitochon-dria. ATP-sensitive K1 (KATP) channels then close, therebytriggering membrane depolarization. The subsequent open-ing of voltage-dependent Ca21 channels leads to an in-crease in [Ca21]i, which is the main trigger for insulinsecretion (16). In the present study, we show that in-creased [Ca21]i is also a key factor in glucose-inducedspreading of rat b-cells on 804G matrix.

RESEARCH DESIGN AND METHODS

Islet isolation and b-cell purification. Islets of Langerhans were isolatedby collagenase digestion of pancreata from male Sprague-Dawley rats (180–200 g wt), followed by Ficoll purification using a modification of previously

From the 1Research Laboratories Louis-Jeantet and the 2Division of ClinicalBiochemistry, University of Geneva, Geneva, Switzerland.

Address correspondence and reprint requests to Domenico Bosco, Labora-toires de Recherche Louis-Jeantet, Centre Medical Universitaire, 1, rueMichel-Servet, 1211 Geneva 4, Switzerland. E-mail: [email protected].

Received for publication 16 August 2000 and accepted in revised form 31January 2001.

BSA, bovine serum albumin; [Ca21]i, intracellular free Ca21 concentration;DMEM, Dulbecco’s minimum essential medium; ECM, extracellular matrix;ER, endoplasmic reticulum; FCS, fetal calf serum; GFP, green fluorescentprotein; IBMX, isobutylmethylxanthine; KATP, ATP-sensitive K1 channels;KRBB, Krebs-Ringer bicarbonate buffer; PKA, protein kinase A; PKC, proteinkinase C; PBS, phosphate-buffered saline; PMA, phorbol 12-myristate 13-acetate; RIA, radioimmunoassay; RHPA, reverse hemolytic plaque assay.

DIABETES, VOL. 50, MAY 2001 1039

described procedures (17,18). For cell preparation, the isolated islets wererinsed 3 times with Mg21- and Ca21-free phosphate-buffered saline (PBS) andresuspended in 1.5 ml of the same buffer containing 0.016% of trypsin (activityagainst casein 1:250) and 0.0066% EDTA (Gibco, Life Technologies, Paisley,Scotland). Digestion (with occasional pipetting) occurred for 6 min at 37°Cand was stopped by the addition of 10-ml ice-cold Krebs-Ringer-bicarbonatebuffer (KRBB), pH 7.4, containing 0.5% bovine serum albumin (BSA), 2.8mmol/l glucose, and 10 mmol/l HEPES. b-cells were then separated fromnon–b-cells by autofluorescence-activated sorting using a FACStar-Plus cellsorter (Becton Dickinson), as previously described (18,19).Cell culture. Sorted b-cells were washed twice in 10–15 ml sterile Dulbecco’sminimum essential medium (DMEM) (Gibco, Life Technologies) containing11.1 mmol/l glucose and 10% heat-inactivated fetal calf serum (FCS) andsupplemented with 110 U/ml penicillin, 110 mg/ml streptomycin, and 50 mg/mlgentamicin. After centrifugation for 10 min at 130g, aliquots of 105 cells wereseeded in nonadherent 60-mm diameter Petri dishes containing 3 ml medium.Cells were then incubated for 20 h at 37°C to allow full recovery of any cellsurface molecules that may have been lost or damaged during islet isolation orcell purification.804G matrix preparation and coating of Petri dishes. 804G matrix wasprepared as previously described (15). Briefly, 804G cells (Desmos, San Diego,CA) were grown in DMEM containing 10% FCS and 5.6 mmol/l glucose. Atconfluence, cells were rinsed and maintained for an additional 3 days in thesame medium. Conditioned medium (hereafter referred to as 804G matrix)was collected and used to coat 35-mm culture Petri dishes. To this end, 60-mlaliquots of 804G matrix were layered at the center of 35-mm culture Petridishes. Dishes were kept in a damp box at 37°C for 18–20 h before beingrinsed three times with sterile water and air-dried. Uncoated Petri dishes wereused as controls.Spreading test. b-cells cultured for 20 h in nonadherent Petri dishes werecentrifuged for 5 min at 130g and resuspended at a density of 106 cells per mlin DMEM containing 10% FCS. The medium was supplemented with eitherglucose (concentration as indicated), KCl (25 mmol/l), glucagon (1 mmol/l),isobutylmethylxanthine (IBMX) (0.5 mmol/l), diazoxide (200 mmol/l), SR-7037(1 mmol/l), or phorbol 12-myristate 13-acetate (PMA) (100 nmol/l). We plated60-ml aliquots of this suspension as droplets at the center of Petri dishescoated with 804G matrix, then incubated them at 37°C. Cell morphology wasgenerally analyzed after 1-h, 3-h, and 24-h incubations. Phase-contrast views ofdifferent fields were photographed after 3 and/or 24 h of incubation. Thepercentage of spreading cells was scored either under microscope or onpictures ($100 cells per condition were analyzed). Data were expressed asmeans 6 SE.

In experiments with thapsigargin, cells were first allowed to attach tocoated dishes. The medium was then replaced with 1 ml medium supple-mented with or without 0.5 mmol/l thapsigargin, allowing us to study the samefields before and after incubation with the drug. Spreading was quantified bymeasuring the area profile of cells on numerized images using the NationalInstitutes of Health image software (version 1.62).

Cultured b-cells transfected with a green fluorescent protein (GFP)-containing vector were recovered with trypsin as described above, rinsed withDMEM, and resuspended in DMEM containing either 2.8 or 16.7 mmol/lglucose. Aliquots of this suspension were plated as droplets at the center ofPetri dishes, coated with 804G matrix, and incubated at 37°C. Cell morphologywas analyzed after 24 h of incubation. The percentage of spreading cells wasanalyzed separately for fluorescent (GFP-expressing) and nonfluorescent(GFP-negative nontransfected) cells.Insulin secretion by radioimmunoassay. After 1.5 or 24 h of incubation,medium (;50 ml) was collected and centrifuged to remove any detached cellsand debris. Aliquots were stored at 220°C for subsequent insulin measure-ment performed by classical radioimmunoassay (RIA). Data were expressedas means 6 SE.b-cell transfection. b-cells cultured for 20 h in nonadherent Petri disheswere centrifuged for 10 min at 130g and resuspended at a density of 106 cellsper ml in DMEM containing 10% FCS and 11.1 mmol/l glucose. We plated 60-mlaliquots of this suspension as droplets at the center of Petri dishes coated with804G matrix and incubated them at 37°C for 3 h. Cells were cotransfected with0.8 mg of the DNA contructs pCMV5-BoNT/E (provided by H. Niemann) and apEGFP vector (Clontech) using the TransFast reagent (Promega, Madison,WI), according to the manufacturer’s instructions. After 48 h, cells were de-tached with trypsin, rinsed with KRBB, and used either for a spreading test ormeasurement of insulin secretion by reverse hemolytic plaque assay (RHPA).Insulin secretion by RHPA. Insulin secretion of transfected b-cells wasassessed by an RHPA, as previously described (20,21,15). Briefly, b-cells werediluted in KRBB (pH 7.4) supplemented with 0.1% BSA and 2.8 mmol/l glucose.Packed sheep erythrocytes (5% v/v) (Behring Institute, Marburg, Germany)previously coated with protein A were then mixed with b-cells, and 50–60 ml

of this preparation were injected in Cunningham’s chambers. After 3 h ofincubation at 37°C, the chambers were first rinsed with KRBB containingeither 2.8 or 16.7 mmol/l glucose and then filled with the same buffer,supplemented with a heat-inactivated (45 min at 56°C) anti-insulin guinea pigserum (1:50 dilution) (22). After 1 h at 37°C, chambers were rinsed with KRBBcontaining 2.8 mmol/l glucose, filled with the same buffer containing guineapig complement (1:40) (Behring Institute), and incubated at 37°C for 1 h.Chambers were then filled with a 0.04% (wt/v) solution of trypan blue inKRBB, rinsed with KRBB, and filled with 4% paraformaldehyde.

Analysis was restricted to single cells that excluded trypan blue at the endof the plaque assay. Fluorescent (GFP-positive) cells and nonfluorescent(GFP-negative) cells were analyzed separately. Results are expressed aspercentage of cells surrounded by an hemolytic plaque (20).Degranulation of b-cells. To maximally reduce their insulin content, b-cellswere attached to 804G-coated Petri dishes and incubated for 6 to 12 h inDMEM supplemented with a cocktail of secretagogues (16.7 mmol/l glucose,100 nmol/l PMA, 1 mmol/l IBMX, and 10 mmol/l forskolin). After trypsinization,cells were rinsed with DMEM, resuspended in DMEM containing either 2.8 or16.7 mmol/l glucose, and spreading was analyzed.Immunofluorescence. b-cells were fixed using Bouin’s solution. After 24 h,cells were rinsed three times with PBS, dehydrated and permeabilized withgraded concentrations of ethanol, and incubated for 2 h at room temperaturewith an anti-insulin antibody (Linco) diluted 1:1,000 in PBS. After rinsing,slides were incubated for 1 h at room temperature with a fluorescein-labeledgoat anti–guinea pig second antibody (1:400) (Jackson Immuno ResearchLaboratories). After rinsing in PBS, slides were covered with 0.02% p-phenylenediamine in PBS-glycerol (1:2, v/v) and screened by fluorescencemicroscopy.

RESULTS

Basic conditions of b-cell spreading and secretion in

response to glucose. High glucose concentrations (16.7or 22.2 mmol/l) induce spreading of b-cells attached to804G matrix, as previously reported (15). Cells rapidlydevelop lamellipodia, and spreading becomes readily ap-parent for a number of cells by 3 h and for most of them at24 h (Fig. 1B and Table 1). However, spreading did notoccur when b-cells were cultured on plastic (not shown)or in the absence of a stimulatory concentration of glucose(2.8 mmol/l) (Fig. 1A, Table 1), even after 24 h, suggesting

FIG. 1. Effect of secretagogues on b-cell spreading. b-cells wereattached on 804G matrix and incubated for 24 h with 2.8 mmol/lglucose, 22.2 mmol/l glucose, 0.5 mmol/l IBMX, or 25 mmol/l KCl. Underbasal conditions (A), cells remained rounded. Upon stimulation witheither glucose (B), IBMX (C), or KCl (D), spreading was induced.Pictures are representative fields of at least three independent exper-iments. Bar, 30 mmol/l.

CYTOSOLIC CALCIUM AND b-CELL ADHESION

1040 DIABETES, VOL. 50, MAY 2001

synergism between secretagogues and signals from theextracellular matrix. Insulin measured in the incubationmedium (Fig. 2A) confirmed good responsiveness of thesecells to glucose.Contribution played by protein kinases A and C to

b-cell spreading. Results from the current study (Fig. 1C,Table 1) and those of a previous study (15) show thatIBMX, which stimulates insulin secretion consequent toelevation of cAMP, stimulates b-cell spreading, suggest-ing the contribution of protein kinase A (PKA) in b-cellspreading. This hypothesis is supported by the effect ofglucagon (1 mmol/l), which induced spreading preventedby the PKA inhibitor Rp-cAMP (0.5 mmol/l) (Fig. 3C and D,Table 1). However, Rp-cAMP had no effect on glucose-induced b-cell spreading (Fig. 3B, Table 1), which could

have been expected because the studies by Persaud et al.(23) showed that in b-cells, PKA is neither activated byglucose nor involved in glucose-stimulated insulin secre-tion.

The possibility that IBMX increases [Ca21]i by releasingcalcium from endoplasmic reticulum (ER) has also beentested. To this end, b-cells attached to 804G matrix wereincubated for 1 h with 1 mmol/l thapsigargin to depleteCa21 from intracellular stores (24), then cells were incu-bated in the presence 0.5 mmol/l IBMX. The experimentshowed that after 1–3 h, IBMX-induced spreading wasidentical in both control and thapsigargin-treated b-cells(data not shown).

The role of protein kinase C (PKC) in b-cell spreadingwas studied by incubating cells in the presence of 100

TABLE 1Effect of secretagogues and inhibitors on cell spreading

Percentage of spreading cellsControl SR-7037 Diazoxide Rp-cAMP

2.8 mmol/l glucose 1.6 6 1.6 (5) — — —22.2 mmol/l glucose 88.0 6 3.9 (6) 8.3 6 6.9 (3) 3.8 6 2.4 (4) 95 (1)0.5 mmol/l IBMX 97.8 6 1.2 (5) 23 6 1.5 (3) 49.3 6 16.7 (3) —25 mmol/l KCl 87 6 1.7 (2) 0 (1) — —1 mmol/l glucagon 49 (1) — — 2 (1)

Values are means 6 SEM. Values in parentheses are the number of experiments. In each experiment, $100 cell were analyzed.

FIG. 2. Insulin secretion in the presence of various secretagogues and channel blockers. Total b-cell insulin secretion measured byradioimmunoassay at the end of a 24-h (A–E) or 1.5-h (F) incubation with various secretagogues in the presence or absence of different inhibitors:A: Insulin secretion was increased with a high glucose concentration (22.2 vs. 2.8 mmol/l). B: Insulin secretion of cells incubated with 2.8 mmol/lglucose (2.8 mmol/l) was increased by the addition of 25 mmol/l KCl, although not to the same extent as with 22.2 mmol/l glucose. C: In thepresence of diazoxide, insulin secretion of cells incubated with 22.2 mmol/l glucose was inhibited, whereas secretion of cells incubated with 2.8mmol/l glucose was not affected, and secretion of cells incubated with IBMX was only slightly decreased. D: In the presence of SR-7037, insulinsecretion of cells incubated with 22.2 mmol/l glucose was inhibited, whereas the drug had no effect on the secretion of cells incubated with 2.8mmol/l glucose or with IBMX. E and F: Insulin secretion of cells incubated for 3 h with 2.8 mmol/l glucose was increased by the addition of 100nmol/l PMA to the same extent as with 16.7 mmol/l glucose (F); the effect of PMA became almost undetectable by 24 h (E).

D. BOSCO AND ASSOCIATES


nmol/l PMA. Within 1–3 h of incubation at low glucose (2.8mmol/l) with 100 nmol/l PMA (Fig. 4B), spreading wasincreased to the same extent as in response to 16.7 mmol/lglucose (Fig. 4C). However, after 24 h of incubation in thepresence of PMA, spreading had not augmented (Fig. 4E),in contrast to the progressive spreading induced by 16.7mmol/l glucose (Fig. 4F). This is attributed to the knowndepletion of PKC after long-term exposure to PMA. Like-wise, the insulin response to PMA was similar to 16.7mmol/l glucose after 90 min of stimulation (Fig. 2F), but itwas significantly lower at 24 h (Fig. 2E). To analyze therole of PKC in glucose-induced spreading, b-cells weretreated for 24 h with 500 nmol/l PMA to downregulatePKC, then shifted to 16.7 mmol/l glucose (in the absence or

presence of PMA). Under these conditions, b-cells spreadout within 3 or 24 h, as did the cells incubated in theabsence of PMA (not shown). These results suggest thatPKA or PKC pathways are not necessary for glucose-induced b-cell spreading.Ca21 influx caused b-cell spreading. The results pre-sented above suggest that at least three different pathways(glucose, PKA, and PKC) converge to invoke b-cell spread-ing. To determine whether b-cell spreading was dependenton Ca21 influx, a high concentration of KCl was used todepolarize cells, thus opening L-type Ca21 channels andraising [Ca21]i (25,16). The results show that in the pres-ence of 2.8 mmol/l glucose, 25 mmol/l KCl was able topromote cell spreading as efficiently as did other secreta-gogues (Fig. 1D, Table 1). This effect was observed at both3 and 18 h of incubation. Insulin secretion, as expected,was also stimulated by KCl (Fig. 2B).Effect of diazoxide and SR-7037 on glucose- and

IBMX-induced b-cell spreading. To further assess theputative role of Ca21 influx in b-cell spreading, we usedthe sulfonamide diazoxide (which activates KATP chan-nels), thus impairing both glucose-induced depolarizationof the membrane and the entry of calcium ions. As shownin Fig. 5 and Table 1, 200 mmol/l diazoxide added to cellsincubated in the presence of 22.2 mmol/l glucose inhibitedspreading. However, the agent only slightly affectedspreading induced by 0.5 mmol/l IBMX (Fig. 5E), whichcould have been expected because cAMP and PKA acti-vate calcium entry downstream of the KATP channels. Aspredicted, glucose-induced insulin secretion was inhibitedin the presence of diazoxide, whereas IBMX-induced insu-lin secretion decreased only slightly (Fig. 2C). Theseresults support the hypothesis that increased [Ca21]i rep-resents a necessary step in secretagogue-induced b-cellspreading.

To further address the role of calcium influx through theL-type voltage-gated Ca21 channels in b-cell spreading,L-type Ca21 channels were blocked with the specific agentSR-7037 (belfosdil). As shown in Fig. 5C and Table 1, 1

FIG. 3. Glucagon-induced spreading is prevented by PKA inhibition.b-cells attached on 804G matrix were incubated either with 16.7 mmol/lglucose (A and B) or with 2.8 mmol/l glucose supplemented with 1mmol/l glucagon (C and D), in the absence (A and C) or in the presenceof Rp-cAMP (B and D), respectively. Incubation was carried out for24 h. Spreading induced by glucagon (C) was markedly decreased byRp-cAMP (D), whereas spreading induced by glucose (A) was notsignificantly affected by the inhibitor (B). Pictures are representativefields of three independent experiments. Bar, 30 mm.

FIG. 4. Effect of PMA on b-cell spreading. b-cells attached on 804Gmatrix were incubated with 2.8 mmol/l glucose in the absence (A andD) or presence of 100 nmol/l PMA (B and E), and with 16.7 mmol/lglucose (C and F). Incubation was carried out for 3 h (A, B, and C) or24 h (D, E, and F). By 3 h, spreading of b-cells was greatly increased inthe presence of PMA, to a larger extent than with high levels ofglucose. At 24 h, spreading did not evolve in the presence of PMA, andit significantly progressed in the presence of 16.7 mmol/l glucose.Phase-contrast views represent representative fields of three indepen-dent experiments. Bar, 30 mm.

FIG. 5. Effects of a KATP channel activator (diazoxide) and a calcium-channel blocker (SR-7037) on b-cell spreading. b-cells attached on804G matrix were incubated for 24 h with 22.2 mmol/l glucose (A, B,and C) or with 0.5 mmol/l IBMX (D, E, and F) in the absence (A and D)or presence of either diazoxide (B and E) or SR-7037 (C and F).Diazoxide completely inhibited spreading induced by glucose (B) andpartially decreased spreading induced by IBMX (E). SR-7037 inhibitedspreading induced by either glucose (C) or IBMX (F). Pictures arerepresentative fields of at least three independent experiments. Bar,30 mm.



mmol/l SR-7037 abolished b-cell spreading induced by 22.2mmol/l glucose and decreased the spreading induced by0.5 mmol/l IBMX (Fig. 5F). As expected, KCl-inducedspreading was also inhibited by SR-7037 (Table 1). In allconditions, the effect was already visible by 3 h of incuba-tion. In the presence of the Ca21 channel blocker, insulinresponse to glucose was greatly decreased, whereas theresponse to IBMX was only slightly diminished (Fig. 2D).Reversibility of b-cell spreading was inhibited by

thapsigargin. b-Cells that had spread regained a roundmorphology when shifted from high (22.7 mmol/l) to low(2.8 mmol/l) glucose concentrations. The effect was de-tected by 3 h, and it extended to all cells within 24 h. Todetermine whether the reticular Ca21 pump that dissipateselevation of [Ca21]i was involved in the reversibility ofspreading induced by glucose, the specific pump inhibitorthapsigargin was added to culture medium concomitantlywith the switch to low glucose. The results showed thatthe reversibility of spreading was prevented at both 3 hand 24 h by 0.5 mmol/l thapsigargin (Fig. 6). When thapsi-gargin was added to round b-cells incubated at 2.8 mmol/lglucose, spreading was not observed at 3 h and increasedonly slightly after 24 h (data not shown).b-cell spreading is not related to exocytotic events.

All conditions tested suggested that increased [Ca21]i is aprerequisite to b-cell spreading. However, increased[Ca21]i is also associated with increased insulin secretion,raising the possibility that spreading is in fact secondary toexocytotic processes. To try to resolve this conundrum,three approaches were used.

First, exogenous insulin was added at different concen-trations (from 1 to 20 mg/ml) to the incubation medium,producing no effect on b-cell spreading at either 2.8 or 22.2mmol/l glucose when compared with cells without addedinsulin. Furthermore, conditioned medium collected after

a 24-h incubation of 2 3 106 b-cells per ml at 2.8 mmol/lglucose failed to induce spreading of cells seeded at 2 3105 cells per ml, suggesting that spreading was not affectedby insulin or other factors constitutively released by cells.

Second, b-cells were degranulated by a 12- to 24-hincubation in the presence of 16.7 mmol/l glucose supple-mented with 100 nmol/l PMA, 10 mmol/l forskolin, and 0.5mmol/l IBMX. Cells were then trypsinized and attached onpetri dishes coated with 804G matrix. Under these condi-tions, they were still able to maximally spread out inresponse to 16.7 mmol/l glucose (Fig. 7).

Finally, sorted b-cells were transfected with botulinumneurotoxin E (BoNT/E), which is known to hydrolyze a25-kDa synaptosome-associated protein, leading to inhibi-tion of exocytosis (26,27). Transfected b-cells were taggedby cotransfection with a GFP vector. Insulin secretion oftransfected cells was then analyzed using an RHPA (Fig.8A and B). Following a 1-h stimulation with 16.7 mmol/lglucose, 9% (7 of 78, total of three different experiments)of GFP-positive cells were surrounded by a hemolyticplaque, compared with 75% of GFP-negative cells analyzedin the same preparation, indicating that secretion wasinhibited in most transfected cells. Nevertheless, after 18 hof incubation on 804G matrix in the presence of 16.7mmol/l glucose (Fig. 8C–H), 50% of 100 GFP-labeled cellswere spread (n 5 2) in the exact same proportion (50%) asobserved for nontransfected cells.

These results strongly suggest that spreading of b-cellsrequires increased [Ca21]i, but is not dependent on theconsequence of increased insulin secretion.

DISCUSSION

In previous studies, we showed that rat islet b-cells spreadin response to glucose when attached on the matrix

FIG. 6. Reversibility of glucose-induced spreading was prevented by thapsigargin. b-cells attached on 804G matrix were incubated in the presenceof 16.7 mmol/l (A and B) or 2.8 mmol/l glucose for 24 h (C), and an initial microscopic evaluation was made (0 h). Then, b-cells incubated with16.7 mmol/l glucose were exposed to 2.8 mmol/l glucose either in the absence (A) or the presence of thapsigargin (B). Cells first incubated with2.8 mmol/l glucose were then exposed to 16.7 mmol/l glucose (C). Pictures of the same fields were taken after 3 and 24 h of incubation. Bar graphsrepresent the quantitative measurements of the results: ;100 cells were measured for each interval and expressed as percentage of maximumspreading. Spread b-cells regained a round morphology when shifted from a high to low glucose concentration (A). This reversibility wasabolished in the presence of thapsigargin (B).



produced by a rat bladder carcinoma cell line (804G) (15).Furthermore, in a mixed population of cells, it was ob-served that spread cells secrete more insulin acutely inresponse to glucose compared with cells that remainrounded. These results suggest bi-directional signalingbetween the islet b-cell and the extracellular matrix.

The primary purpose of the present study was to getinsight into possible intracellular pathways linking glucosestimulation, b-cell spreading, and insulin secretion. Theexperiments demonstrate that a sustained rise in [Ca21]i isrequired for glucose-induced rat b-cell spreading on 804Gmatrix. The effect is mimicked by any maneuver whichraises [Ca21]i (IBMX, KCl, PMA) and is prevented byblocking L-type calcium channels (Belfosdil). Diazoxide,which activates KATP channels, also prevented glucose-mediated spreading and secretion.

The effect of PKA and PKC activation on [Ca21] is still amatter of controversy, particularly in the absence ofglucose. However, both PKA (28) and PKC (29,30) havebeen shown to activate Ca21 currents and/or L-type cal-

cium channels in control conditions. Even though PKAand PKC increase both [Ca21]i and insulin secretion, it isthought that the [Ca21]i increase induced by these kinasesis not necessary for insulin release (28,29). This does notexclude the possibility that a [Ca21] increase after PMAand IBMX treatment can be involved in cell spreading. Inour study, the effect of IBMX was only partially blocked bydiazoxide, probably because PKA activates calcium entrydownstream of the KATP channels. However, because theeffect is also partially blocked by SR7037, we cannotexclude that PKA-induced spreading could be partiallyindependent of [Ca21]. The possibility that IBMX increases[Ca21] by releasing calcium from ER has been tested. Theexperiment showed that IBMX induced the same extent ofspreading in both control and thapsigargin-treated b-cells,suggesting that IBMX does not induce spreading by releas-ing Ca21 from the ER.

Although both PKA or PKC were shown to increasespreading, an obligatory role for one or the other inglucose-induced spreading is unlikely because the effect ofglucose persists in the absence of activity of the twoenzymes. In fact, our results show that after long-termPMA treatment (PKC downregulation) and in the presenceof an inhibitor of PKA (RpcAMP), glucose is still able toinduce spreading of b-cells. In this work, the mechanismsby which PKA and PKC activate spreading have not beeninvestigated beyond the possible role in glucose-inducedspreading.

Spreading of b-cells in response to glucose is probablyrelated to glucose metabolism and not to an autocrineeffect secondary to granule exocytosis because it was notabolished in degranulated cells. That the effect of glucoseis not related to exocytosis is supported by the observa-

FIG. 7. Insulin immunofluorescence of degranulated b-cells induced tospread by glucose. Indirect immunofluorescence of b-cells first ex-posed for 6 h in the presence of 16.7 mmol/l glucose, 1 mmol/l IMBM,100 nmol/l PMA, and 10 mmol/l forskolin (degranulating cocktail) thencultured for 3 h with 16.7 mmol/l glucose on 804G matrix. At the end ofthe experiment, cells were fixed, permeabilized, immunostained forinsulin (B), and stained with Evan’s blue to show cell profile (A). Inthese spread cells, labeling is restricted to the perinuclear region(Golgi area). No significant labeling is observed in other cytoplasmicregions. Of the total cell population, ;30% had similar morphology andlabeling. Bar, 20 mm.

FIG. 8. Inhibition of exocytosis by transfection of botulinus toxin didnot affect glucose-induced b-cell spreading. A and B: Insulin secretion(detected by RHPA) of b-cells cotransfected with GFP and BoNT/E.Phase-contrast (A) and the corresponding fluorescent view (B) revealfour b-cells that are negative for GFP and surrounded by a hemolyticplaque and one GFP-positive b-cell (arrow) that is not surrounded bya hemolytic plaque. C– H: Phase contrast (C, E, and G) and correspond-ing fluorescent views (D, F, and H) of b-cells cotransfected for GFPand BoNT/E attached to 804G matrix and incubated for 24 h with 16.7mmol/l glucose. Both GFP-positive cells (i.e., toxin-positive) and GFP-negative cells (toxin-negative) were able to spread. For quantitativeanalysis of the data, see text. Bar, 20 mm.



tion that it persists in b-cells transfected with botulinustoxin, which prevents secretion by .90%, as previouslyshown (31) and confirmed in the current study. Theseexperiments also indicate that spreading is not the resultof presentation of adhesion molecules at the cell surfaceduring exocytosis.

The present results suggest a signaling pathway bywhich glucose-increased [Ca21]i modulates cell-matrix in-teractions (inside-out signaling). A role of [Ca21]i in inside-out signaling has been described previously. A correlationbetween intracellular calcium and integrin-mediated celladhesion has been reported in other cell types (32–35).Cytosolic calcium modulates integrin affinity (36–38), andprevention of a rise in [Ca21]i rise alters integrin-mediatedadhesion of neutrophils (39,40). Whether calcium actsthrough changes in integrin conformation and/or by post-receptor events, such as integrin-cytoskeleton association,is not completely understood. The cytoplasmic domain ofintegrins is the target of cytoplasmic modulators of inte-grin affinity (41). Ca21-binding proteins that associate withintegrins include calreticulin, calcineurin, calpain, andcalmodulin, which are all present within b-cells. Studiesare now under way to assess whether they are involved inglucose-mediated b-cell spreading. A rise in [Ca21]i maynot be a consequence of spreading in response to glucosebecause a rise in [Ca21]i in response to glucose has beenobserved in b-cells in suspension (42), and it has beenroutinely observed in b-cells attached to plastic or glass,on which they do not spread.

The cell surface molecules responsible for transducingcell matrix signaling in b-cells is unknown. We and othershave reported the presence of b1 integrins on rat andhuman b-cells. Interestingly, it has been found that a6b1,but not a3b1, expression is increased at the surface ofb-cells in response to glucose, and anti-a6 antibodiesgreatly reduce b-cell spreading (15); thus the hypothesiswas that integrin a6b1 might be the b-cell transmembraneprotein responsible for cell-matrix signaling by glucose.

Our previous studies showed a correlation betweenspreading and insulin secretion of individual cells (out-side-in signaling). Interestingly, calcium influx is alsoknown to increase in response to endothelial cell spread-ing (36,38,43). This suggests a positive feedback mecha-nism involving the extracellular matrix and resulting inimproved insulin response to glucose. An increased [Ca21]i inresponse to a6b1 ligation would support this hypothesis.

In conclusion, the present study demonstrated a crucialrole for calcium influx through the L-type voltage-gatedCa21 channels in rat islet b-cell spreading in response tosecretagogues, including glucose (inside-out signaling).Further studies are needed to determine to what extentthe induced spreading plays a role in the regulation ofsecretion, and whether increased [Ca21]i is also involvedin outside-in signaling.

ACKNOWLEDGMENTS

This work was supported by Grant no. 1–1998-213 fromthe Juvenile Diabetes Foundation International (D.G.R).Additional support was provided by the Swiss DiabetesAssociation (D.G.R.); the Raymond Berger Foundation,Lausanne, Switzerland (D.G.R.); and Swiss National Sci-

ence Foundation grants 32–50776.97 (D.G.R.), 32–49755.96(C.B.W.), and 31–50811.97 (P.A.H.).

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