A Key Role for β-Cell Cytosolic Phospholipase A2 in the Maintenance of Insulin Stores But Not in the Initiation of Insulin Secretion

Diabetes, Jan 2002

Cytosolic phospholipase A2 (cPLA2) is a Ca2+-sensitive enzyme that has been implicated in insulin secretion in response to agents that elevate β-cell intracellular Ca2+ ([Ca2+]i). We generated clones of the MIN6 β-cell line that stably underexpress cPLA2 by transfection with a vector in which cPLA2 cDNA had been inserted in the antisense orientation. Reduced expression of cPLA2 was confirmed by Western blotting. The insulin content of cPLA2-deficient MIN6 cells was reduced by ∼90%, but they showed no decrease in preproinsulin mRNA expression. Measurements of stimulus-dependent changes in [Ca2+]i indicated that reduced expression of cPLA2 did not affect the capacity of MIN6 cells to show elevations in Ca2+ in response to depolarizing stimuli. Perifusion experiments indicated that cPLA2 underexpressing MIN6 pseudoislets responded to glucose, tolbutamide, and KCl with insulin secretory profiles similar to those of cPLA2 expressing pseudoislets, but that secretion was not maintained with continued stimulus. Analysis of the ultrastructure of cPLA2-deficient MIN6 cells by electron microscopy revealed that they contained very few mature insulin secretory granules, but there was an abundance of non–electron-dense vesicles. These data are consistent with a role for cPLA2 in the maintenance of insulin stores, but they suggest that it is not required for the initiation of insulin secretion from β-cells.

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A Key Role for β-Cell Cytosolic Phospholipase A2 in the Maintenance of Insulin Stores But Not in the Initiation of Insulin Secretion

A Key Role for -Cell Cytosolic Phospholipase A in the 2 Maintenance of Insulin Stores But Not in the Initiation of Insulin Secretion Shanta J. Persaud 1 3 Helen M. Roderigo-Milne 1 3 Paul E. Squires 1 2 David Sugden 1 3 Caroline P.D. Wheeler-Jones 0 1 Phil J. Marsh 1 4 Ve´ ronique D. Belin 1 3 Melanie J. Luther 1 3 Peter M. Jones 1 3 0 Veterinary Basic Sciences, Royal Veterinary College , London , U.K. 1 RESEARCH DESIGN AND METHODS Materials. MIN6 cells were obtained from Dr. Y. Oka and Professor J.-I. Miyazaki (University of Tokyo , Tokyo , Japan). cPLA 2 sequence on a 1.6-kbp fragment. This was gel-purified and ligated to pcDNA3.1 that had 3 Endocrinology and Reproduction Research Group, King's College London , London , U.K. 4 Molecular Biology Unit, King's College London, London, U.K. 3.2A, New Hunt's House, King's College London , London SE1 1UL, U.K 5 kit was from Dynal (Oslo , Norway). Restriction endonucleases were obtained from Promega (Madison, WI), and pcDNA3.1 was from Stratagene Europe (Amsterdam , the Nether- lands). PCR primers were prepared in-house (Molecular Biology Unit, King's College London), and real-time quantitative PCR was performed using a LightCycler rapid thermal cycler system from Roche Diagnostics (Lewes, Sussex, U.K.). General laboratory chemicals , including fura-2 acetoxymethyl- ester (fura-2/AM), 3-aminopropyltriethoxysilane (APES), forskolin, and phor- bol myristate acetate (PMA) were purchased from Sigma (Poole, Dorset , U.K.) . The Axiovert 135 Research Inverted microscope was obtained from Carl Zeiss (Welwyn Garden City, U.K.), the Axon Imaging Workbench was from Axon Instruments (Foster City, CA), and the ISIS camera was from Photonics Science (Roberts-Bridge, Sussex, U.K.). Vector construction, transfection of MIN6 cells, and selection of clones. Antisense expression constructs were made by digesting pSG5 to completion with BglII and partially with EcoRI to yield the cPLA Cytosolic phospholipase A2 (cPLA2) is a Ca2 -sensitive enzyme that has been implicated in insulin secretion in response to agents that elevate -cell intracellular Ca2 ([Ca2 ]i). We generated clones of the MIN6 -cell line that stably underexpress cPLA2 by transfection with a vector in which cPLA2 cDNA had been inserted in the antisense orientation. Reduced expression of cPLA2 was confirmed by Western blotting. The insulin content of cPLA2-deficient MIN6 cells was reduced by 90%, but they showed no decrease in preproinsulin mRNA expression. Measurements of stimulus-dependent changes in [Ca2 ]i indicated that reduced expression of cPLA2 did not affect the capacity of MIN6 cells to show elevations in Ca2 in response to depolarizing stimuli. Perifusion experiments indicated that cPLA2 underexpressing MIN6 pseudoislets responded to glucose, tolbutamide, and KCl with insulin secretory profiles similar to those of cPLA2 expressing pseudoislets, but that secretion was not maintained with continued stimulus. Analysis of the ultrastructure of cPLA2-deficient MIN6 cells by electron microscopy revealed that they contained very few mature insulin secretory granules, but there was an abundance of non- electron-dense vesicles. These data are consistent with a role for cPLA2 in the maintenance of insulin stores, but they suggest that it is not required for the initiation of insulin secretion from -cells. Diabetes 51:98 -104, 2002 - Cmasses 85 kDa (cPLA2 ), 114 kDa (cPLA2 ), ytosolic phospholipase A2 (cPLA2) is a family of enzymes encoding proteins of molecular and 61 kDa (cPLA2 ), all of which selectively hydrolyze membrane phospholipids to generate arachidonic acid ( 1 ). The and forms of cPLA2 are sensitive to free Ca2 levels reached in stimulated cells, whereas cPLA2 is Ca2 -independent ( 1 ). To date, most studies have focused on the physiological roles played by cPLA2 , the first member of the cPLA2 family to be identified, and it has been implicated in a variety of cellular processes, including mitogenesis, allergic responses, fertility, and cytotoxicity (reviewed in references 2 and 3). cPLA2 has been identified in islets and -cells by Western blotting, Northern blotting, and polymerase chain reaction (PCR) ( 4 – 6 ), and the sensitivity of -cell cPLA2 to Ca2 in the micromolar range ( 7 ) places it as a potential effector downstream of the well-defined initial stages in -cell stimulus-secretion coupling, which result in increases in intracellular Ca2 . Experimental evidence supportive of a role for cPLA2 in insulin secretion has been provided using inhibitors of the enzyme ( 8 ), and it has long been known that arachidonic acid is an effective stimulator of insulin secretion ( 9 ). In the current study, we addressed the function of cPLA2 in -cells by generating -cell lines in which cPLA2 is permanently underexpressed by stable transfection of the MIN6 insulin-secreting -cell line with a vector encoding cPLA2 in the antisense orientation. This approach has allowed us to demonstrate that -cells deficient in cPLA2 1) can mount an appropriate insulin secretory response, but that secretion is not maintained, and 2) show a reduced capacity to synthesize and store insulin. been cut with EcoRI and BamHI. The DNA was prepared for transfection by banding plasmid preparations on CsCl according to standard methods. MIN6 cells, grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 15% fetal calf serum, 100 units/ml penicillin, 100 g/ml streptomycin, and 2 mmol/l glutamine, were electroporated (2 kV/cm, 3 F) with HindIIIlinearized cPLA2 antisense vector or with linearized cDNA3.1 to which cPLA2 antisense cDNA had not been ligated (empty vector). Neo-resistant transfected cells were selected by growth in medium supplemented with 1 mg/ml G418. Colonies of resistant cells were expanded for analysis and functional studies. Western blotting. Extracts of G418-resistant -cell clones were prepared by sonication in a lysis buffer ( 10 ), and protein concentration was determined by the Bradford method ( 11 ) using bovine serum albumin as standard. Protein (200 g) was fractionated on 10% polyacrylamide gels and transferred to polyvinylidene difluoride membranes. cPLA2 protein was detected by an enhanced chemiluminescence system using an anti-cPLA2 rabbit polyclonal primary antibody (1:5,000 dilution) and an anti-rabbit IgG conjugated with horseradish peroxidase (1:10,000 dilution). Insulin secretion. MIN6 cells in monolayers do not show robust secretory responses to physiological stimulators of insulin secretion, but their secretory output improves when they are configured as three-dimensional aggregates, which we term “pseudoislets” (PIs) ( 12 ). To maximize secretory performance, we therefore carried out all secretion experiments using MIN6 PIs. Initial experiments were performed in static incubations, where groups of 10 PIs were incubated for 1 h in the presence of a physiological salt solution ( 13 ) supplemented with agents of interest, and insulin secretion was measured by radioimmunoassay ( 14 ). For all additional secretion experiments, dynamic insulin release was assessed using a multichamber perifusion system at 37°C in a temperature-controlled environment, essentially as described for rat islets ( 15 ). Ca2 microfluorimetry. MIN6 cells were seeded onto APES-coated glass coverslips and allowed to adhere overnight in DMEM under standard tissue culture conditions. Agonist-evoked changes in cytosolic Ca2 were examined in MIN6 cells grouped within monolayer clusters (3–20 cells/cluster), rather than from single cells in isolation, which are poorly nutrient-responsive ( 12 ). Cell clusters were loaded for 20 min at 37°C with 2.5 mol/l of the Ca2 fluorophore fura-2/AM. The coverslips were washed and placed in a steel chamber, the volume of which was 500 l. A single 22-mm coverslip formed the base of the chamber, which was mounted into a heating platform on the stage of an Axiovert 135 Research Inverted microscope. All experiments were carried out at 37°C using a Na -rich balanced salt solution as the standard extracellular medium ( 16 ). A low-pressure, rapid superfusion system (flow rate 1–2 ml/min) was used to change the solutions in the bath. Cells were illuminated alternatively at 340 nm and 380 nm using an Axon Imaging Workbench. Emitted light was filtered using a 510-nm long-pass barrier filter and detected using an ISIS camera. Changes in the emission intensity of fura-2 expressed as a ratio of dual excitation were used to indicate changes in [Ca2 ]i using established procedures ( 17 ). Data were collected every 3 s for multiple regions of interest in any one field of view. All records have been corrected for background fluorescence (determined from cell-free coverslip). Preproinsulin mRNA expression. Messenger RNA was isolated from control and cPLA2-deficient MIN6 cells using a Dynabeads Oligo(dT)25 kit according to the manufacturer’s instructions. Oligo(dT)18 (1 g) and random 10-mers (1 g) were added to the mRNA (10 l), and the mixture was heated (70°C, 5 min) to remove secondary RNA structure then cooled on ice. DTT (10 mmol/l), dATP, dCTP, dTTP, and dGTP (all 0.5 mmol/l); recombinant ribonuclease inhibitor (80 , RNAsin); MMLV-RT (200 ); and diethyl pyrocarbonatetreated water were added to give a final volume of 20 l, and the mixture was incubated at 42°C for 50 min. MMLV-RT was inactivated by heating at 70°C for 15 min. The cDNA was diluted 10-fold with tRNA (10 g/ml) and used immediately or stored at 20°C for future use. An aliquot of mRNA was not reverse-transcribed and was diluted with tRNA and stored at 85°C. Mouse preproinsulin I and II cDNA sequences ( 18 ) were used to design forward and reverse PCR primers that detected both preproinsulin mRNAs: sense primer, 5 -AAC CCA CCC AGG CTT TTG TC-3 ; antisense primer, 5 -TGC AGT AGT TCT CCA GCT GG-3 . The predicted size of the preproinsulin PCR product was 267 bp. Forward and reverse glyceraldehyde-3phosphate dehydrogenase (GAPDH) PCR primers were as follows: sense primer, 5 -CCC ATC ACC ATC TTC CAG GAG C-3 ; antisense primer, 5 -CCA GTG AGC TTC CCG TTC AGC-3 ( 19 ). The predicted size of the GAPDH PCR product was 473 bp. PCR products amplified by the preproinsulin and GAPDH primers were separated by agarose gel electrophoresis (2% wt/vol) and visualized by staining with ethidium bromide (0.5 g/ml). The preproinsulin product was cut from the gel and spin column purified using a Qiaquick gel extraction kit, and its identity was confirmed by sequencing on an ABI 377 using fluorescent chain-terminator methods. Concentrations of PCR products were determined FIG. 1. Depletion of -cell cPLA2 expression. MIN6 -cells were transfected with a vector housing cPLA2 in the antisense (AS) orientation, and stably transfected cells were selected by maintenance in medium supplemented with 1 mg/ml G418. The top panel shows cPLA2 expression of three stably transfected clones, termed N, I, and K, compared with passage-matched native MIN6 cells. The decreased expression of cPLA2 was stable, as shown by the blot in the lower panel performed on cell extracts prepared 11 months after the immunoblot shown in the top panel. by densitometry by comparison with known amounts of molecular weight markers. Tenfold serial dilutions were prepared as standards ranging from 1 fg to 100 pg. Real-time PCR amplification was performed using a LightCycler rapid thermal cycler system. Reactions were performed in a 10- l volume containing nucleotides, Taq DNA polymerase, and buffer (all included in the LightCycler-DNA Master SYBR Green I mix); MIN6 cell cDNA; and 3 mmol/l MgCl2 and 0.5 mol/l primers. All PCR protocols included a 10-s denaturation step and then continued for 40 or 45 cycles consisting of a 95°C denaturation for 0 s, annealing for 10 s at 55°C (GAPDH) or 57°C (preproinsulin), and a 72°C extension phase for 11 s (preproinsulin) or 19 s (GAPDH). Fluorescence measurements were taken at 85°C for 2 s to eliminate fluorescence from primer-dimer formation. The amplification products of both primer pairs were subjected to melting point analyses and subsequent gel electrophoresis to ensure specificity of amplification. Electron microscopy. Transmission electron micrographs of glutaraldehydefixed, osmium-stained MIN6 cells were prepared by John Pacy of King’s College London Electron Microscope Unit using standard techniques. Data analysis. Data are expressed, where appropriate, as means SE and were analyzed statistically using Student’s t test, analysis of variance, and Bonferroni’s multiple comparison test as appropriate. Differences between treatments were considered significant at P 0.05. RESULTS Immunodetection of cPLA2. Changes in MIN6 -cell cPLA2 expression after transfection with a vector coding cPLA2 in the antisense orientation was assessed by Western blotting of extracts of G418-resistant MIN6 cell populations. It can be seen from Fig. 1 (top panel) that native MIN6 cells expressed a form of cPLA2 that migrated with an apparent molecular mass of 110 kDa, consistent with the reported retarded mobility of cPLA2 on polyacrylamide gels relative to its predicted molecular mass ( 20 ). G418resistant -cell clones were expanded and harvested after transfection with the antisense cPLA2 plasmid, and Fig. 1 shows that antisense clones, termed I, N, and K, showed much reduced expression of cPLA2. The underexpression was stable, with no recovery of cPLA2 expression with continued passage. This is clearly shown in Fig. 1 (bottom panel), where the loss of cPLA2 expression by clone N was still apparent after continued passaging for 11 months. This clone was used for functional analysis unless otherwise indicated in the text or figure legends. Protein and insulin content of MIN6 cells underexpressing cPLA2. The protein and insulin contents of PIs were measured in parallel with their use in secretion experiments, and comparisons were made between passage-matched, unselected, nontransfected cells (“controls”); MIN6 cells transfected with an empty vector (“EV”); and MIN6 cells transfected with cPLA2 antisense cDNA (“antisense”). Protein content of PIs underexpressing cPLA2 was of a similar level to passage-matched control PIs (antisense N, 262 13.9 ng protein/PI; antisense I, 293 41.7; control, 242 26.2; n 9; P 0.2), but insulin content was considerably lower in the cPLA2deficient PIs (antisense N, 0.15 0.03 ng insulin/PI; antisense I, 0.13 0.05; control, 1.42 0.04; EV, 1.85 0.17; n 5–16; P 0.001, control and EV versus antisense). Insulin secretion from MIN6 cells underexpressing cPLA2. Basal insulin secretion (2 mmol/l glucose) from cPLA2-deficient PIs was significantly less than that from equivalent passage control PIs (control, 0.24 0.02 ng PI 1 h 1; antisense N, 0.02 0.005 ng PI 1 h 1, mean of four separate experiments; P 0.001), consistent with their much reduced insulin content. Insulin secretion measurements in static incubations indicated that although the amount of insulin secreted by cPLA2-depleted PIs was very low, the fold increases in secretion in response to depolarizing stimuli were not significantly different from responses elicited by control cells (20 mmol/l KCl: control, 368 30% basal; antisense N, 417 13% basal; 100 mol/l tolbutamide: control, 197 16% basal; antisense N, 233 62% basal, mean of four separate experiments; P 0.05 versus basal; P 0.1, antisense N versus control). However, the small magnitude of the secretory response of late passage MIN6 cells to glucose precluded meaningful analysis of any possible alterations in nutrient-induced insulin secretion after reduced cPLA2 expression in static incubation experiments. Additional secretion experiments were therefore performed in perifusions in which small changes in secretion could be determined readily, and expression of secretion data as a percentage of basal insulin release allows direct comparison of the profile of secretion from the two populations. It can be seen from Fig. 2 that 20 mmol/l glucose caused a significant increase in insulin secretion from control PIs, with a short-lived peak and a sustained plateau. Despite their much-reduced insulin content and insulin output, cPLA2 underexpressing MIN6 PIs (clones N and I) showed a similar profile of insulin secretion to that seen with control PIs after a glucose challenge (Fig. 2). cPLA2 underexpressing PIs also showed secretory profiles in response to KCl and tolbutamide that were similar to those of control PIs (Fig. 3; clone N). However, cells underexpressing cPLA2 showed a much reduced magnitude of response upon exposure to potentiators of insulin secretion after an initial stimulus with glucose, KCl, or tolbutamide. Thus, activation of protein kinase C by PMA or increases in cyclic AMP in response to forskolin resulted in significant potentiation of insulin secretion from control PIs, but secretory output declined with time after depletion of cPLA2 (Fig. 3). This was not a consequence of defects in signaling systems at the level of effector enzymes because cPLA2-deficient PIs showed robust secretory responses to forskolin when it was added as the primary stimulus, but secretion again declined with continued stimulation (Fig. 4; clone N), suggesting an inability of the cPLA2-deficient cells to maintain a prolonged secretory response. Stimulus-induced changes in intracellular Ca2 in MIN6 cell clusters underexpressing cPLA2. Reduced expression of cPLA2 did not affect Ca2 handling by MIN6 -cells. Thus, resting cytosolic Ca2 was not significantly different (P 0.2) between control and cPLA2-deficient cells (clones N and I), and it can be seen from Fig. 5 that underexpression of cPLA2 had minimal effects on Ca2 signaling in response to nutrients and depolarizing stimuli. Thus, cells derived from clones N and I showed increases in Ca2 in response to nutrients (glucose and KIC) and non-nutrients (tolbutamide and KCl), and these responses were not significantly different from passage-matched control cells in terms of proportion of cells responding. Preproinsulin mRNA expression in cPLA2-deficient -cells. A real-time fluorescence-based PCR method (LightCycler) was used to determine whether the site of the lesion in cPLA2-deficient -cells that resulted in abnormally low insulin content was at the level of preproinsulin mRNA expression. Primers were used to amplify a preproinsulin fragment from cPLA2-deficient -cell clones N and I and passage-matched control MIN6 -cells. Quantification was performed by standardizing preproinsulin product against a sequence-verified preproinsulin fragment, and these values were normalized against the content of GAPDH mRNA in the same extracts. The melting point curves obtained using GAPDH and preproinsulin primers in the LightCycler indicated that single products were amplified (Fig. 6, top panel); the middle panel shows that the products were of the appropriate predicted sizes (267 bp for preproinsulin and 473 bp for GAPDH). The standard FIG. 3. Secretion from cPLA2-deficient -cells is not sustained. MIN6 PIs underexpressing cPLA2 (clone N [Œ]) and passage-matched control, cPLA2-expressing PIs (F) were perifused with buffers supplemented as shown by the horizontal bars. The magnitude of the secretory responses of cPLA2-deficient PIs to depolarizing stimuli (glucose, A; tolbutamide, B; KCl, C) were not significantly different (P > 0.2) from those of control PIs. However, cPLA2-underexpressing MIN6 PIs showed significantly (P < 0.05) reduced secretory responses to PMA (A and B) and forskolin (C) after the initial response to depolarizing stimuli. Data show means of two separate channels for controls, means SE of three separate channels for cPLA2-depleted. PMA-induced insulin secretion from antisense clone I after an initial glucose stimulus was also transient, with secretion during the last 10 min of PMA stimulation being 383 39% basal, compared with 269 75% basal for antisense N (P > 0.2) and 1,074 135% basal for controls (P < 0.01). FIG. 4. Signaling via adenylate cyclase is intact in cPLA2-deficient -cells. MIN6PIs underexpressing cPLA2 (clone N [Œ]) and passagematched control, cPLA2-expressing PIs (F) were perifused with buffers that contained 10 mol/l forskolin at 2 or 20 mmol/l glucose, as shown by the horizontal bars. Both populations of PIs showed a similar secretory profile in response to forskolin at 2 mmol/l glucose. However, control PIs showed a further, sustained increase in secretion after an elevation from 2 to 20 mmol/l glucose, still in the presence of 10 mol/l forskolin, whereas cPLA2-deficient PIs showed a transient secretory response. Data are means of two separate channels for controls and means SE of three separate channels for cPLA2-depleted. curves generated for the two products were linear (Fig. 6, bottom panel), and quantification of preproinsulin mRNA in the samples using these standard curves indicated that cPLA2-deficient -cells contained levels of preproinsulin mRNA that were not significantly different (P 0.2) from passage-matched control cells (control, 16.6 2.8 fg preproinsulin mRNA/fg GAPDH mRNA, n 3; antisense N, 14.0 2.7, n 4; antisense I, 17.9 7.6, n 2). Transmission electron micrographs of MIN6 cells underexpressing cPLA2. The morphology of MIN6 cells underexpressing cPLA2 was consistent with the insulin content data. Thus, it is clear from Fig. 7 that cells derived from antisense clone N had considerably less secretory granules than control cells, and they showed altered morphology of the endoplasmic reticulum and Golgi network, with indication of the formation of non– electrondense vesicle-like organelles. In all other respects, the ultrastructure of the cPLA2-deficient MIN6 cells was indistinguishable from that of native MIN6 cells. An abundance of non– electron-dense vesicles and a scarcity of insulin granules were also observed in micrographs prepared from cells derived from antisense clone I (data not shown). DISCUSSION We and others have shown that the conventional cPLA2 (cPLA2 ) is expressed in islets and -cells ( 4 – 6 ) and that arachidonic acid, the product of cPLA2 activation, is capable of initiating insulin secretion (reviewed in ref. 21). The sensitivity of cPLA2 to concentrations of Ca2 reached in -cells after Ca2 entry in response to nutrientinduced membrane depolarization has made it an attractive candidate as a pivotal transducer of nutrientgenerated signals in -cells, and data obtained using inhibitors of cPLA2 are consistent with its being involved in glucose-induced insulin secretion ( 8 ). However, cPLA2 inhibitors lack specificity ( 3 ), so we have now examined the effects of stable underexpression of cPLA2 on the function of the insulin-secreting -cell line MIN6. We chose to use the MIN6 cell line for three specific reasons. First, we required a cell line rather than primary islets so that we could create stable transfects and expand the transfected populations so that all cells, whether at the single cell level or in population experiments, were deficient in cPLA2. Second, the MIN6 cell line was established by cloning from a pancreatic insulinoma ( 22 ), so clones established after transfection are likely to have been derived from common parental cells. Third, MIN6 cells retain their glucose responsiveness with continued passage when configured as PIs ( 12 ), which was an absolute requirement when assessing nutrient-induced effects of expanded clones. Maintenance of the altered phenotype was assessed by immunodetection of cPLA2 expression in clones soon after selection and again after continued culture of the cells in G418-containing medium for 11 months. These measurements consistently showed that cPLA2 expression was much reduced in the G418-resistant clones. The cPLA2 underexpressing -cells grew on tissue culture plastic as monolayers indistinguishable from cPLA2 expressing control cells, and they formed PIs when plated into gelatin-coated flasks, similar to native MIN6 cells ( 12 ). Functional studies were performed to determine whether depletion of cPLA2 affected the ability of -cells to respond to agents that elevate intracellular Ca2 and might be expected to activate this Ca2 -sensitive enzyme. Our Ca2 microfluorimetry data indicated that depletion of cPLA2 had no effect on the proportion of cells that were able to respond to glucose, KIC, tolbutamide, and KCl or on the amplitudes of the mean basal to peak Ca2 responses. These data are entirely consistent with cPLA2 being a Ca2 -responsive enzyme that can be placed downstream in the stimulus-response coupling cascade to the initial glucose metabolism, membrane depolarization, and Ca2 influx events. Thus, it is clear that loss of cPLA2 expression does not affect the capacity of -cells to rdeecpooglnarizizee acnedllsm,oertapbeorlmiziet tghlueceonster,y colofsCea2KA.TP channels, Measurement of the insulin content of cPLA2-deficient PIs revealed that they had only 10% of the content of control cells. The significantly reduced rate of basal insulin secretion by MIN6 cells underexpressing cPLA2 was consistent with their greatly reduced insulin content. However, the depleted insulin content did not markedly affect the capacity of cPLA2-deficient cells to respond to nutrient and non-nutrient secretagogues and to depolarizing stimuli with increases in insulin secretion. Thus, when insulin secretion was expressed relative to the appropriate basal levels of secretion, cPLA2-deficient PIs showed initial secretory profiles that were not significantly different from those obtained with control, cPLA2-containing PIs. However, whereas control PIs showed sustained insulin secretory responses, secretion from -cells underexpressing cPLA2 was not maintained when an initial stimulus was followed by further stimulation by activators of protein kinase C or A. Experiments in which cPLA2-depleted PIs were initially exposed to forskolin indicated that PKA activity was not compromised and that the inability of the cells to maintain insulin output was independent of the stimulus. The most likely explanation for this, given the reduced insulin content of the cPLA2 underexpressing -cells, is that their intracellular stores of insulin were sufficient for an initial secretory response but that they soon became depleted and secretion therefore declined with continued stimulus. Morphological analysis by electron microscopy was consistent with this interpretation, and the micrographs indicated two distinct features of MIN6 cells underexpressing cPLA2. First, they showed a significant reduction in the number of secretory granules per cell that was considerably greater than the natural decline in insulin secretory granule number that occurs with continued passaging of MIN6 cells. Second, and distinct from anything we have observed with prolonged culture of control MIN6 cells, was the increased incidence of non– electrondense vesicles within cPLA2-deficient cells. This profound change in the ultrastructure of cells underexpressing cPLA2 did not seem to have a generalized deleterious effect on protein synthesis by these -cells, because their protein content was not reduced compared with controls, but their insulin content was reduced by 90%. A recent study in which cPLA2 was overexpressed in kidney epithelial cells ( 23 ) implicated cPLA2 in intracellular vesicle trafficking processes, and the data obtained in the current study are consistent with cPLA2 being required for appropriate architecture of the Golgi apparatus. Disruption of the trans-Golgi network would affect the formation of secretory vesicles, resulting in decreased insulin content despite maintained insulin mRNA levels, as was observed in our experiments. In summary, the current data indicate that cPLA2 is not required for pancreatic -cells to show an initial secretory response to nutrient and non-nutrient secretagogues, suggesting that it is not a pivotal Ca2 -sensitive sensor in -cell stimulus-secretion coupling. However, our data are consistent with a role for cPLA2 in the maintained secretory response, perhaps as a consequence of its role in Golgi budding to generate insulin secretory granules. ACKNOWLEDGMENTS We are grateful to Diabetes U.K. for project grant support (RD97/0001525). P.E.S. was an RD Lawrence Fellow of Diabetes UK (RD97/0001453). V.D.B. is funded by the Eli Lilly International Foundation. We thank Dr. Y. Oka and Professor J.I. Miyazaki (University of Tokyo, Tokyo, Japan) for provision of the MIN6 cells, Dr. B. Kennedy (Merck Frosst, PQ, Canada) for the cPLA2 vector, and Dr. R. Kramer (Lilly, Indianapolis, IN) for the cPLA2 antiserum. 1. 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Shanta J. Persaud, Helen M. Roderigo-Milne, Paul E. Squires, David Sugden, Caroline P.D. Wheeler-Jones, Phil J. Marsh, Véronique D. Belin, Melanie J. Luther, Peter M. Jones. A Key Role for β-Cell Cytosolic Phospholipase A2 in the Maintenance of Insulin Stores But Not in the Initiation of Insulin Secretion, Diabetes, 2002, 98-104, DOI: 10.2337/diabetes.51.1.98