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J. Biol. Chem., Vol. 276, Issue 16, 13198-13208, April 20, 2001

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Studies of Insulin Secretory Responses and of Arachidonic Acid Incorporation into Phospholipids of Stably Transfected Insulinoma Cells That Overexpress Group VIA Phospholipase A₂ (iPLA₂) Indicate a Signaling Rather Than a Housekeeping Role for iPLA₂^*

Zhongmin Ma, Sasanka Ramanadham, Mary Wohltmann, Alan Bohrer, Fong-Fu Hsu, and John Turk

From the Mass Spectrometry Resource, Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110

Received for publication, November 16, 2000, and in revised form, January 2, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A cytosolic 84-kDa group VIA phospholipase A₂ (iPLA₂) that does not require Ca²⁺ for catalysis has been cloned from several sources, including rat and human pancreatic islet -cells and murine P388D1 cells. Many potential iPLA₂ functions have been proposed, including a signaling role in -cell insulin secretion and a role in generating lysophosphatidylcholine acceptors for arachidonic acid incorporation into P388D1 cell phosphatidylcholine (PC). Proposals for iPLA₂ function rest in part on effects of inhibiting iPLA₂ activity with a bromoenol lactone (BEL) suicide substrate, but BEL also inhibits phosphatidate phosphohydrolase-1 and a group VIB phospholipase A₂. Manipulation of iPLA₂ expression by molecular biologic means is an alternative approach to study iPLA₂ functions, and we have used a retroviral construct containing iPLA₂ cDNA to prepare two INS-1 insulinoma cell clonal lines that stably overexpress iPLA₂. Compared with parental INS-1 cells or cells transfected with empty vector, both iPLA₂-overexpressing lines exhibit amplified insulin secretory responses to glucose and cAMP-elevating agents, and BEL substantially attenuates stimulated secretion. Electrospray ionization mass spectrometric analyses of arachidonic acid incorporation into INS-1 cell PC indicate that neither overexpression nor inhibition of iPLA₂ affects the rate or extent of this process in INS-1 cells. Immunocytofluorescence studies with antibodies directed against iPLA₂ indicate that cAMP-elevating agents increase perinuclear fluorescence in INS-1 cells, suggesting that iPLA₂ associates with nuclei. These studies are more consistent with a signaling than with a housekeeping role for iPLA₂ in insulin-secreting -cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Phospholipases A₂ (PLA₂)¹ catalyze hydrolysis of the sn-2 fatty acid substituent from glycerophospholipid substrates to yield a free fatty acid and a 2-lysophospholipid (1-7). PLA₂ are a diverse group of enzymes, and the first members to be well characterized have low molecular masses (~14 kDa), require millimolar [Ca²⁺] for catalytic activity, and function as extracellular secreted enzymes designated sPLA₂ (3, 6). The first PLA₂ to be cloned that is active at [Ca²⁺] that can be achieved in the cytosol of living cells is an 85-kDa protein classified as a group IV PLA₂ and designated cPLA₂ (3, 5). This enzyme is induced to associate with its substrates in membranes by rises in cytosolic [Ca²⁺] within the range achieved in cells stimulated by extracellular signals that induce Ca²⁺ release from intracellular sequestration sites or Ca²⁺ entry from the extracellular space, is also regulated by phosphorylation, and prefers substrates with sn-2 arachidonoyl residues (5).

A second cytosolic PLA₂ has been cloned (8-10) that does not require Ca²⁺ for catalysis, and it is classified as a group VIA PLA₂ and has been designated iPLA₂ (3, 4). The iPLA₂ enzymes cloned from hamster (8), mouse (9), and rat (10) cells represent species homologs, and all are 84-kDa proteins containing 752 amino acid residues with highly homologous sequences. Each contains a GXSXG lipase consensus motif and eight stretches of a repeating motif homologous to a repetitive motif in the integral membrane protein-binding domain of ankyrin (8-10). Each of these iPLA₂ enzymes is susceptible to inhibition (8-10) by a bromoenol lactone (BEL) suicide substrate (11, 12) that is not an effective inhibitor of sPLA₂ or cPLA₂ enzymes at comparable concentrations (4, 11-14). It has been proposed that this enzyme now be designated iPLA₂ to distinguish it from a membrane-associated, Ca²⁺-independent PLA₂ that contains a peroxisomal targeting sequence and is designated iPLA₂ (15, 16).

Proposed functions for iPLA₂ include a housekeeping role in phospholipid remodeling that involves generation of lysophospholipid acceptors for incorporation of arachidonic acid into phospholipids of murine P388D1 macrophage-like cells (4, 17, 18). This proposal (4) derives from experiments involving inhibition of iPLA₂ activity in P388D1 cells with BEL (17) or with an antisense oligonucleotide (18). Inhibition of P388D1 cell iPLA₂ activity suppresses incorporation of [³H]arachidonic acid into phospholipids and reduces [³H]lysophosphatidylcholine (LPC) levels in [³H]choline-labeled cells (17, 18). Arachidonate incorporation (17, 18) reflects a deacylation/reacylation cycle (19, 20) of phospholipid remodeling rather than de novo synthesis (21), and the level of LPC acceptors is thought to limit the rate of [³H]arachidonic acid incorporation into P388D1 cell phosphatidylcholine (PC) (17, 18).

Many other potential iPLA₂ functions have been proposed (22-54), and the facts that multiple splice variants are differentially expressed among cells and form hetero-oligomers with distinct properties suggest that iPLA₂ gene products might have multiple functions (23, 31, 44, 45). Proposed iPLA₂ functions include signaling in secretion (10, 22, 25, 29, 48-50), and we and others (47-54) have found that, in pancreatic islets, BEL attenuates glucose-induced insulin secretion, arachidonate release, and rises in islet -cell cytosolic [Ca²⁺]. Both pancreatic islets and brain contain electrically active secretory cells that express high levels of iPLA₂ (10), and iPLA₂ is the vastly predominant brain cytosolic PLA₂ (24, 34, 35). BEL also inhibits iPLA₂ (15) and phosphatidate phosphohydrolase-1 (PAPH-1) (55), and the ambiguity of pharmacologic studies makes manipulating iPLA₂ expression by molecular biologic means an attractive alternative to study iPLA₂ functions. We report here the preparation of two stably transfected insulinoma cell lines that overexpress iPLA₂. We have studied insulin secretory responses, arachidonate incorporation into phosphatidylcholine, and iPLA₂ subcellular location in these lines.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- ECL detection reagents and 1-palmitoyl-2-[¹⁴C]linoleoyl-sn-glycero-3-phosphocholine (55 mCi/mmol) were purchased from Amersham Pharmacia Biotech. Phosphatidylcholine standards were obtained from Avanti Polar Lipids (Birmingham, AL) and arachidonic acid from Nu-Chek Prep (Elysian, MN). BEL iPLA₂ suicide substrate (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one was purchased from Cayman Chemical (Ann Arbor, MI). Tissue culture media (CMRL-1066, RPMI, and minimal essential medium), penicillin, streptomycin, Hanks' balanced salt solution, and L-glutamine were purchased from Life Technologies, Inc. Fetal bovine serum was obtained from HyClone (Logan, UT) and Pentex bovine serum albumin (BSA, fatty acid-free, fraction V) from ICN Biomedical (Aurora, OH). ATP, ampicillin, IBMX, propranolol, and kanamycin were obtained from Sigma and forskolin from Calbiochem (La Jolla, CA). Krebs-Ringer bicarbonate buffer (KRB) contained 25 mM HEPES, pH 7.4, 115 mM NaCl, 24 mM NaHCO₃, 5 mM KCl, 1 mM MgCl₂.

Cell Culture-- INS-1 insulinoma cells provided by Dr. Christopher Newgard (University of Texas, Dallas, TX) were cultured as described (56-58) in RPMI 1640 medium containing 11 mM glucose, 10% fetal calf serum, 10 mM Hepes buffer, 2 mM glutamine, 1 mM sodium pyruvate, 50 mM -mercaptoethanol, 100 units/ml penicillin, and 100 µg/ml streptomycin. RetroPack PT 67 cells (CLONTECH, Palo Alto, CA) were maintained in Dulbecco's modified Eagle's medium (4.5 mg/ml glucose) containing 10% fetal bovine serum, 4 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin.

Preparation of Recombinant Retrovirus Containing the cDNA Encoding the Rat Pancreatic Islet iPLA₂-- A retroviral system (59, 60) was used to stably transfect INS-1 cells with iPLA₂ cDNA and achieve overexpression. To construct the retroviral vector, iPLA₂ cDNA (10) was subcloned into EcoRI-BglII multiple cloning sites of pMSCVneo vector using the CLONTECH murine stem cell retrovirus (MSCV) expression system. Full-length rat pancreatic islet iPLA₂ cDNA was excised from pBK-CMV-iPLA₂ vector and subcloned into the retroviral vector pMSCVneo at the recognition sites for restriction endonucleases EcoRI and XhoI. The construct containing the iPLA₂ cDNA (pMSCVneo-iPLA₂) was transfected into CLONTECH RetroPack PT 67 packaging cells with a GenePORTER transfection system according to the manufacturer's instructions (Gene Therapy Systems, San Diego, CA). Upon transfection of packaging cells, pMSCVneo integrated into the genome and expressed a transcript containing viral packaging signal, a neomycin resistance gene that confers resistance to the selection agent G418, and iPLA₂ cDNA. This transcript is recognized by viral proteins in packaging cells. Introduction of pMSCVneo-iPLA₂ into PT 67 cells results in production of high titer, replication-incompetent infectious virus particles that were released into the culture medium, collected, and used to infect INS-1 cells.

Infection of INS-1 Cells with Recombinant Retroviral Particles and Selection of Stably Transfected Cells That Overexpress iPLA₂-- INS-1 cells were plated on 100-mm Petri dishes at a density of 3-5 × 10⁵ cells/plate 12-18 h before infection. Freshly collected, retrovirus-containing medium was passed through a 0.45-µm filter and added to INS-1 cell monolayers. Polybrene (final concentration 4 µg/ml) was added to culture medium, and medium was replaced after 24 h of incubation. To select stably transfected cells that expressed high levels of iPLA₂, retrovirally infected cells were cultured with G418 (0.4 mg/ml) for 1-2 weeks. After G418-resistant colonies became apparent, cell culture was continued for several days. Individual colonies were transferred to a 48-well plate for expansion of clonal cells. Two iPLA₂-overexpressing (iPLA₂-X) lines were obtained that exhibit similar properties not shared by parental cells or clonal lines selected after transfection with empty vector.

Assay of INS-1 Cell iPLA₂ Activity-- Seeded INS-1 were washed with phosphate-buffered saline (PBS) and detached by trituration. Cells were collected by centrifugation and disrupted by sonication (Vibra Cell High Intensity Processor, five 1-s pulses, amplitude 12%) in homogenization buffer (250 mM sucrose, 40 mM Tris-HCI, pH 7.1, 4 °C). Homogenates were centrifuged (15,000 × g, 45 min, 4 °C) to yield a cytosolic supernatant. Protein content was measured with Coomassie regent (Pierce) against bovine serum albumin standard. Ca²⁺-independent PLA₂ activity in aliquots of cytosol (25 µg of protein) was assayed by ethanolic injection (5 µl) of 1-palmitoyl-2-[¹⁴C]linoleoyl-sn-glycero-3-phosphocholine (final concentration 5 µM) in assay buffer (40 mM Tris, pH 7.5, 5 mM EGTA; total volume 200 µl). Assay mixtures were incubated (3 min, 37 °C, with shaking) and reactions terminated by adding butanol (0.1 ml) and vortexing. After centrifugation (2,000 × g, 5 min), products in the butanol layer were analyzed by silica gel G TLC in petroleum ether/ethyl ether/acetic acid (80/20/1). The TLC plate region containing free fatty acid was identified with iodine vapor and scraped into a scintillation vial. Released [¹⁴C]fatty acid was measured by liquid scintillation spectrometry, and PLA₂ specific activity was calculated from dpm of released fatty acid and protein content as described (61).

Immunoblotting Analyses of INS-1 Cell iPLA₂ Protein-- INS-1 cell cytosolic proteins were analyzed by SDS-PAGE and transferred to a nylon membrane that was subsequently blocked (3 h, room temperature) with Tris-buffered saline plus Tween (TBS-T, 20 mM Tris-HCl, 137 mM NaCl, pH 7.6, 0.05% Tween 20) containing 5% milk protein. The blot was then washed (TBS-T, 5 min, five times) and incubated (1 h, room temperature) with a polyclonal antibody (1:2000 dilution in TBS-T) to iPLA₂ generated by multiple antigen core technology against peptides in the iPLA₂ deduced amino acid sequence, as described below. The nylon membrane was then washed in TBS-T (5 min, five times) and incubated (1 h, room temperature) with a secondary antibody coupled to horseradish peroxidase (Roche Molecular Biochemicals) at 1:40,000 dilution in TBS-T containing 3% BSA. The antibody complex was visualized by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech).

Determination of Insulin Secretion by INS-1 Insulinoma Cells-- Culture medium from INS-1 cells seeded in 24-well plates was removed, and the cells were washed twice in KRB medium and incubated (1 h, 37 °C, under an atmosphere of 95% air, 5% CO₂) in KRB medium (1 ml). Medium was then removed and replaced with KRB medium containing glucose (0-18 mM) with or without forskolin (2.5 µM), IBMX (100 µM), or dibutyryl cAMP (1 mM). In experiments with BEL (10 µM) or propranolol (250 µM), these agents were added both to preincubation medium and to medium for the experimental incubation. After addition of final incubation medium, cells were incubated (1 h, 37 °C) under the atmosphere described above, and medium was then removed for measurement of insulin by radioimmunoassay (62). Cells were then detached from the plate and their acid-ethanol extractable insulin determined by radioimmunoassay (63). Secreted insulin was expressed as a fraction of total cellular insulin content (64).

Determination of INS-1 Cell cAMP Content-- After experimental incubations, medium was removed from each well, and ice-cold ethanol (0.4 ml) containing IBMX (100 µM) was added. After a 5-min room temperature incubation, cells were detached with a rubber policeman, and ethanol and cells were placed in glass test tubes (12 × 75 mm). Cells were sedimented by centrifugation (1500 × g, 10 min), and the supernatant was placed in a clean test tube, concentrated to dryness under nitrogen, and reconstituted in 50 mM phosphate buffer (0.4 ml). The cAMP content was measured by enzyme immunoassay and normalized to cell protein content (65-67).

Incubation of INS-1 Cells with Arachidonic Acid to Induce Phospholipid Remodeling-- INS-1 cells were cultured in RPMI medium containing penicillin, streptomycin, fungizone, and gentamicin (0.1% w/v each). Cells (1.2 × 10⁶/condition) were treated (30 min, 37 °C) with vehicle only or with BEL (10 µM). Medium was then removed and replaced with fresh medium containing no supplements other than those described above or containing arachidonic acid (final concentration 70 µM), and cells were cultured at 37 °C. After 0, 2, 6, 8, or 24 h, cells were washed twice with PBS, suspended in homogenization buffer, and disrupted by sonication. Lipids were extracted (68) and the extract concentrated and analyzed by NP-HPLC.

Chromatographic Analyses of Phospholipids-- Phospholipid head-group classes were separated by NP-HPLC (47) analyses on a silicic acid column (LiChrosphere Si-100 (10 µm, 250 × 4.5 mm); Alltech, Deerfield, IL) with the solvent system hexane, 2-propanol, 25 mM potassium phosphate, pH 7.0, ethanol, acetic acid (367/490/62/100/0.6) at a flow of 0.5 ml/min for 60 min and then 1.0 ml/min. The retention time of standard PC was 102 min.

Electrospray Ionization Mass Spectrometric Analyses of Choline-containing Lipids-- PC species were analyzed as Li⁺ adducts by ESI/MS on a Finnigan (San Jose, CA) TSQ-7000 triple stage quadrupole mass spectrometer with an ESI source controlled by Finnigan ICIS software. Phospholipids were dissolved in methanol/chloroform (9/1, v/v) containing LiOH (2 nmol/µl), infused (1 µl/min) with a Harvard syringe pump, and analyzed under described conditions (69, 70). For tandem MS, precursor ions selected in the first quadrupole were accelerated (32-36 eV collision energy) into a chamber containing argon (2.3-2.5 millitorr) to induce collisional-activated dissociation, and product ions were analyzed in the final quadrupole to identify PC species in the total ion current profile (70).

Measurement of Nonesterified Arachidonic Acid in INS-1 Cells by Isotope Dilution Gas Chromatography Negative Ion Electron Capture Mass Spectrometry-- Parental and iPLA₂-overexpressing INS-1 cells that had been incubated with supplemental arachidonic acid for 24 h as described above were washed with 0.1% BSA in KRB four times to remove unincorporated arachidonic acid and were then preincubated for 30 min in KRB medium containing 10 µM BEL or BEL-free vehicle. The precincubation medium was then removed, and the cells were incubated for 1 h at 37 °C in KRB medium containing 0.1% BSA, 2.5 mM CaCl₂, and 2 or 11 mM glucose without or with IBMX (100 µM). Incubations were terminated by extraction with 2 ml of chloroform/methanol (1/1) containing 330 pmol of [²H₈]arachidonic acid internal standard. Extracts were analyzed by RP-HPLC to isolate arachidonic acid, which was converted to a pentafluorobenzyl ester derivative and analyzed by GC/MS in negative ion electron capture mode (47). Selected monitoring of carboxylate anions of arachidonate (m/z 303) and [²H₈]arachidonate (m/z 311) was performed to quantitate arachidonate by reference to a standard curve (47). The amount of arachidonic acid was expressed as a ratio to the lipid phosphorus content of the extract.

Immunocytofluorescence Localization of iPLA₂ within INS-1 Cells-- We prepared an iPLA₂ antibody by multiple antigen core methods (Research Genetics) that link eight peptide copies to an octameric lysine core (71). The iPLA₂ peptides coupled to this core for immunizing rabbits were ²⁵KEVSLADYASSERVRE⁴¹ and ⁴⁸⁹RMKDEVFRGSRPY⁵⁰¹. In INS-1 cells that overexpress iPLA₂, this antibody recognizes an 84-kDa protein corresponding to full-length iPLA₂ (Fig. 1), and recognition of the protein is blocked by including the immunizing peptides in incubations with the antibody. To determine the subcellular location of iPLA₂, cells were allowed to attach to chambered glass slides overnight and then treated with experimental solutions. At the end of incubations, cells were rinsed in PBS, fixed in 4% paraformaldehyde, washed with PBS, fixed in ice-cold methanol, washed, and blocked in a PBS solution containing globulin-free BSA (1%), Triton X-100 (0.3%), and goat serum (3%). Primary antibodies (either preimmune immunoglobulin or anti-iPLA₂, 0.003 µg/ml) were then added, and cells were incubated (18 h, 4 °C) in a humidified chamber. Cells were then washed and incubated (1 h, in the dark) with a secondary antibody (affinity-purified goat anti-rabbit IgG, 1:200 dilution) coupled to the fluorophore Cy3. Cells were then washed and covered with Antifade solution (Molecular Probes, Eugene, OR). Slides were mounted with coverslips and examined by confocal microscopy at excitation and emission wavelengths of 550 and 570 nm, respectively.

Statistical Analyses-- Student's t test was used to compare two groups, and multiple groups were compared by one-way analysis of variance with post hoc Newman-Keul's analyses.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Transfection of INS-1 insulinoma cells with a retroviral construct containing the rat iPLA₂ cDNA followed by selection of G418-resistant cells resulted in the isolation of two stably transfected clones that expressed severalfold more iPLA₂ activity than parental INS-1 cells (Fig. 1A). Like the iPLA₂ activity in pancreatic islets and in other insulinoma cell lines (10), the iPLA₂ activity in the stably transfected cells was stimulated by 1 mM ATP and virtually completely inhibited by 10 µM BEL (Fig. 1A). The iPLA₂-X cell lines also exhibited an increased content of an 84-kDa protein that was recognized by an iPLA₂ antibody after SDS-PAGE and immunoblotting analyses (Fig. 1B). Increased iPLA₂ expression was a stable property of the transfected cells and persisted on serial passage in culture.

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Overexpression of iPLA2 in INS-1 insulinoma cells stably transfected with iPLA2 cDNA in a retroviral vector. Two clonal INS-1 cell lines were prepared by stable transfection with a retroviral vector containing iPLA2 cDNA as described under "Experimental Procedures," and levels of their iPLA2 activity (panel A) and immunoreactive protein (panel B) were compared with those of parental INS-1 cells. PLA2 activity was measured in the absence of Ca2+ and presence of EGTA without (open bars) or with 1 mM ATP alone (cross-hatched bars) or ATP and 10 µM BEL (solid bars). The leftmost set of bars reflects activity from parental cells (control) and the center and rightmost sets of bars reflect activity from the two clonal INS-1 cell lines transfected with iPLA2 cDNA in the retroviral construct (#1-iPLA2-X and #2-iPLA2-X, respectively). Immunoblotting analyses (panel B) were performed after SDS-PAGE analyses of INS-1 cell cytosolic protein. After transfer to nylon membranes, proteins were probed with an iPLA2 antibody and visualized with ECL as described under "Experimental Procedures." Migration positions of molecular size markers are illustrated at the left margin of panel B, and the three experimental lanes represent immunoreactive proteins from parental INS-1 cells (control) and the two iPLA2-overexpressing lines (#1-iPLA2-X and #2-iPLA2-X), respectively. The arrow at the right indicates the expected migration position of an 84-kDa protein corresponding to full-length rat iPLA2.

When treated with the adenylyl cyclase activator forskolin (2.5 µM), the iPLA₂-X INS-1 cells secreted more insulin than did parental INS-1 cells or INS-1 cells transfected with an empty retroviral construct that did not contain iPLA₂ cDNA (Fig. 2). The magnitude of this effect increased with the medium glucose concentration over the range 0-5 mM. Similar responses to glucose and forskolin were observed with both of the iPLA₂-X cell lines (data not shown). Similarly, both of the iPLA₂-X cell lines exhibited increased insulin secretory responses, compared with control INS-1 cells, when stimulated with the cAMP phosphodiesterase inhibitor IBMX, and this response also increased with the medium glucose concentration over the range 0-5 mM (Fig. 3). The magnitude of the increased insulin secretory response to glucose was similar when either forskolin or IBMX was used as the cAMP-elevating stimulus, and the combination of forskolin and IBMX induced only slightly greater secretory responses than either agent alone (data not shown).

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Effects of glucose and the adenylyl cyclase activator forskolin on insulin secretion from iPLA2-overexpressing and control INS-1 cells. Insulin secretion was measured after 1-h incubation of INS-1 cells in medium containing 0, 2, or 5 mM glucose without or with 2.5 µM forskolin and normalized to cell insulin content to yield fractional secretion values. Secretory responses were compared among parental INS-1 cells, INS-1 cells transfected with an empty retroviral vector without iPLA2 cDNA, and stably transfected INS-1 cells that overexpressed iPLA2 (clone 1 from Fig. 1). Mean fractional secretion values are displayed, and S.E. are indicated (n = 6). Values for forskolin-treated iPLA2-X INS-1 cells were significantly higher (p < 0.05) than those from non-forskolin-treated cells and than those from control cells at all [glucose].

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Effects of glucose and the cAMP phosphodiesterase inhibitor IBMX on insulin secretion from iPLA2-overexpressing and control INS-1 cells. Secretion studies were similar to those in Fig. 2 except that IBMX (0.1 mM) and not forskolin was used to elevate cAMP. Mean fractional secretion values are displayed and S.E. indicated (n = 6). Values for IBMX-treated iPLA2-X cells were significantly higher (p < 0.05) than those for non-IBMX-treated cells and than those for control cells at glucose concentrations of 2 and 5 mM.

Increasing the medium glucose concentration above 5 mM did not further amplify the insulin secretory response to forskolin (Fig. 4A) or IBMX (Fig. 4B) above that achieved with 5 mM glucose and either forskolin or IBMX for iPLA₂-X INS-1 cells. Unlike control INS-1 cells, the iPLA₂-X cells exhibited enhanced insulin secretion in response to either forskolin or IBMX at 0 or 2 mM glucose. With control cells, secretory responses to forskolin or IBMX required the presence of a glucose concentration exceeding 2 mM, and no increase in secretion was induced by those agents at 0 or 2 mM glucose. Purified native -cells isolated by fluorescence-activated cell sorting also fail to secrete insulin in response to glucose alone but exhibit glucose-induced insulin secretion in the presence of cAMP-elevating agents (72), and INS-1 and other insulinoma cell lines exhibit more robust insulin secretory responses to glucose in the presence of cAMP-elevating agents (73, 74).

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Glucose-concentration dependence of effects of forskolin or IBMX to increase insulin secretion from iPLA2-overexpressing and control INS-1 cells. Secretion studies in panels A and B were similar to those in Figs. 2 and 3, respectively, except that glucose concentrations of 8, 11, and 17 mM were also examined. Mean fractional secretion values are displayed and S.E. indicated (n = 6). Values for forskolin- or IBMX-treated iPLA2-X cells were significantly higher (p < 0.05) than those for nontreated cells and than those for control cells at all [glucose].

Both forskolin and IBMX alone and in combination induced an increase in INS-1 cell cAMP content (Fig. 5). Despite the greater insulin secretory responses to cAMP-elevating agents by iPLA₂-X cells compared with control INS-1 cells, iPLA₂-X cells did not exhibit greater rises in cAMP than control cells when stimulated with forskolin or IBMX. This indicates that augmented cAMP accumulation does not explain enhanced insulin secretion by iPLA₂-X cells and suggests that responsiveness of the secretory apparatus to cAMP is increased by iPLA₂ overexpression.

View larger version (31K): [in this window] [in a new window]   Fig. 5.   Effects of glucose, forskolin, and IBMX on cAMP content of iPLA2-overexpressing and control INS-1 cells. The cAMP content was measured by enzyme immunoassay as described under "Experimental Procedures" in parental INS-1 cells (control) or iPLA2-overexpressing INS-1 cells (iPLA2-X, clone 1, Fig. 1) that had been incubated for 1 h without or with forskolin (2.5 µM) or IBMX (0.1 mM) alone or in combination. Mean values for cAMP normalized to cellular protein content are displayed, and S.E. are indicated (n = 6).

At a concentration of 10 µM, the iPLA₂ inhibitor BEL (11, 12) attenuated insulin secretion induced by glucose and IBMX from iPLA₂-X cells and virtually completely suppressed stimulated insulin secretion from control INS-1 cells (Fig. 6A). Similar effects of BEL were observed with cells stimulated with forskolin and glucose (data not shown). At 10 µM, BEL inhibits iPLA₂ activity in iPLA₂-X INS-1 cells by over 95% (Fig. 1). These findings suggest that iPLA₂ participates in acute signaling events involved in induction of insulin secretion by glucose and cAMP-elevating agents. It is of interest that suppression of insulin secretion in response to glucose and cAMP-elevating agents is virtually completely suppressed by BEL in control INS-1 cells but only partially suppressed in iPLA₂-X INS-1 cells. This suggests that the history of iPLA₂ overexpression modifies secretory responsiveness in a manner that is not reversed by acute iPLA₂ inhibition.

View larger version (36K): [in this window] [in a new window]   Fig. 6.   Effects of the iPLA2 suicide substrate BEL and of the phosphatidate phosphohydrolase inhibitor propranolol on insulin secretion induced by glucose and forskolin or IBMX from control and iPLA2-overexpressing INS-1 cells. Panel A, insulin secretion studies were similar to those in Fig. 3, except that the glucose concentration was either 2, 8, or 11 mM, and cells were pre-treated (30 min) with either BEL (10 µM) or with BEL-free vehicle before the experimental incubation. Panel B, insulin secretion studies were similar to those in panel A, except that forskolin (2.5 µM) rather than IBMX (0.1 mM) was used to elevate cAMP, and cells were pre-treated with propranolol (250 µM) or propranolol-free vehicle rather than with BEL before the experimental incubation. Mean fractional secretion values are displayed, and S.E. are indicated (n = 6). BEL-treated cells exhibited significantly (p < 0.05) lower fractional secretion values than non-BEL-treated cells under all tested conditions. Propranolol-treated cells exhibited significantly higher fractional secretion values at 2 and 11 mM glucose for iPLA2-X INS-1 cells.

BEL also inhibits PAPH-1 (55), and effects of BEL are often compared with those of the PAPH inhibitor propranolol to distinguish effects attributable to inhibition of iPLA₂ or to PAPH-1 (47, 75-77). At a concentration that maximally inhibits PAPH in -cells (78) and in amniotic WISH cells (76), propranolol did not mimic the effect of BEL to suppress insulin secretion from iPLA₂-X or control INS-1 cells induced by forskolin and glucose but tended to amplify the secretory response (Fig. 6B). This is consistent with a report that propranolol amplifies insulin secretion from -cells (78). These findings indicate that the suppression by BEL of insulin secretion from iPLA₂-X and control INS-1 cells stimulated with glucose and cAMP-elevating agents is not attributable to inhibition of PAPH-1.

Although BEL partially suppresses stimulated insulin secretion from iPLA₂-X cells, the fact that BEL-treated iPLA₂-X cells continued to secrete more insulin than control INS-1 cells when stimulated with glucose and cAMP-elevating agents (Fig. 6A) suggests that the history of iPLA₂ overexpression altered the secretory responsiveness of the cells in a manner that was not completely reversed by acute inhibition of iPLA₂ activity. It was considered possible that this reflected an effect of iPLA₂ overexpression on INS-1 cell membrane phospholipid composition. Native islet -cells are highly enriched in arachidonate-containing phospholipids compared with cells from other tissues, and such phospholipid species may be important in secretion (69). A housekeeping role for iPLA₂ in generating LPC acceptors for arachidonate incorporation into PC in murine P388D1 cells has been suggested from observations that inhibiting iPLA₂ reduces both P388D1 cell LPC levels and [³H]arachidonate incorporation into PC (4, 17, 18). This suggests that iPLA₂ overexpression might cause increases in INS-1 cell arachidonate-containing phospholipids and secretory responsiveness.

To test this possibility, we examined PC molecular species in iPLA₂-X cells and in parental INS-1 cells by ESI/MS. Fig. 7A is an ESI/MS spectrum of Li⁺ adducts of parental INS-1 cell PC species that had been isolated by NP-HPLC, and it is virtually identical to the PC spectrum obtained from iPLA₂-X INS-1 cells (Fig. 9A). Major ions in the spectrum represent Li⁺ adducts of 16:0/16:1-GPC (m/z 738), 16:1/18:1-GPC (m/z 764), 16:0/18:1-GPC (m/z 766), and 18:1/18:1-GPC (m/z 792). The identities of the species represented by these ions were determined by collisionally activated dissociation and tandem MS (70). Arachidonate-containing species at m/z 788 (16:0/20:4-GPC), m/z 816 (18:0/20:4-GPC), or m/z 814 (18:1/20:4-GPC) are not abundant. Adding supplemental arachidonic acid to the culture medium caused the INS-1 cells to remodel their phospholipids in a time-dependent fashion so that the abundance of arachidonate-containing PC species increased. After 6 h, signals for 16:0/20:4-GPC, 18:1/20:4-GPC, and 18:0/20:4-GPC had increased severalfold (Fig. 7B), and by 18 h, 16:0/20:4-GPC had become the most abundant PC species in the mixture (Fig. 7C). The iPLA₂-X INS-1 cells incorporated arachidonic acid into PC (Fig. 7D) in a manner similar to that of parental INS-1 cells, and the time course of appearance of arachidonate-containing PC species was virtually identical for iPLA₂-X and control INS-1 cells (Fig. 8).

View larger version (41K): [in this window] [in a new window]   Fig. 7.   Electrospray ionization mass spectrometric analyses of glycerophosphocholine lipid species in iPLA2-overexpressing and control INS-1 cells incubated with supplemental arachidonic acid. Control (panels A-C) or iPLA2-overexpressing (panel D) INS-1 cells were incubated in medium with supplemental arachidonic acid for 0 h (panel A), 6 h (panel B), or 18 h (panels C and D). Lipids were then extracted and analyzed by NP-HPLC to isolate GPC lipids, which were analyzed by ESI/MS as Li+ adducts. Identities of the GPC lipid species represented by ions in the total ion current profile were determined by collisionally activated dissociation and tandem MS.

View larger version (26K): [in this window] [in a new window]   Fig. 8.   Time course of appearance of arachidonate-containing GPC lipid species in control and iPLA2-overexpressing INS-1 cells incubated with supplemental arachidonic acid. Experiments were performed as in Fig. 7, and ion current intensities at m/z 788 (16:0/20:4-GPC), 816 (18:0/20:4-GPC), and 814 (18:1/20:4-GPC) were expressed as a ratio to that at m/z 766 (16:0/18:1-GPC). Mean values are displayed, and S.E. are indicated (n = 4).

After treatment of -cells with BEL, iPLA₂ activity recovers slowly, and there is virtually no measurable activity for 6 h (47). To determine whether iPLA₂ inhibition alters the early time course of arachidonate incorporation into INS-1 cell PC, appearance of arachidonate-containing PC species was compared in non-BEL-treated and in BEL-treated iPLA₂-X INS-1 cells (Fig. 9). In non-BEL-treated cells incubated with supplemental arachidonic acid, the abundance of 16:0/20:4-GPC and of 18:0/20:4-GPC increased between time 0 (Fig. 9A) and 2 h of incubation (Fig. 9B) and increased further at 6 h (Fig. 9C). A similar response occurred in BEL-treated iPLA₂-X INS-1 cells (Fig. 9D), and the time course of appearance of arachidonate-containing PC species was virtually identical in non-BEL-treated and in BEL-treated iPLA₂-X INS-1 cells (Fig. 10). Similar results were obtained with parental INS-1 cells (data not shown). Findings in Figs. 7-10 do not support a role for iPLA₂ in arachidonic acid incorporation into INS-1 cell PC, even under conditions where the enzyme is overexpressed.

View larger version (42K): [in this window] [in a new window]   Fig. 9.   ESI/MS analyses of glycerophosphocholine lipid species in iPLA2-overexpressing INS-1 cells treated with BEL or BEL-free vehicle and then incubated with supplemental arachidonic acid. iPLA2-overexpressing INS-1 cells were treated with BEL (panel D) or with BEL-free vehicle (panels A-C) and then incubated in culture medium with supplemental arachidonic acid for 0 h (panel A), 2 h (panel B), or 6 h (panels C and D). Lipids were extracted and analyzed by NP-HPLC and ESI/MS as in Fig. 7.

View larger version (30K): [in this window] [in a new window]   Fig. 10.   Time course of appearance of arachidonate-containing GPC lipid species in iPLA2-overexpressing INS-1 cells that had been treated with BEL or with BEL-free vehicle and then incubated with supplemental arachidonic acid. Experiments were performed as in Fig. 9 and data analyzed as in Fig. 8. Mean values are displayed, and S.E. are indicated (n = 4).

When the nonesterified arachidonate content of iPLA₂-overexpressing INS-1 cells was measured by isotope dilution GC/MS using [²H₈]arachidonic acid as internal standard (Fig. 11), values for cells incubated with 11 mM glucose without or with 100 µM IBMX were 18.9 and 27.1 pmol/nmol of lipid phosphorus, respectively. Nonesterified arachidonate levels were 6.7 pmol/nmol of lipid phosphorus higher for cells incubated with 11 mM glucose than for those incubated with 2 mM glucose, and arachidonate release was suppressed to 27% of control values when the cells were treated with BEL. No net increase in nonesterified arachidonate content was observed for parental INS-1 cells upon increasing the medium glucose concentration or adding IBMX to the incubations, although BEL reduced the basal content of nonesterified arachidonate to 21% of control values.

View larger version (28K): [in this window] [in a new window]   Fig. 11.   Isotope dilution gas chromatographic mass spectrometric quantitation of nonesterified arachidonate content of iPLA2-overexpressing INS-1 cells. Stably transfected iPLA2-overexpressing INS-1 cells were treated with BEL-free vehicle (panels A and B) or with 10 µM BEL for 30 min, and medium was then removed. Cells were placed in fresh incubation medium containing glucose at a concentration of 2 mM (panel A) or 11 mM (panels B and C) and incubated for 1 h as under "Experimental Procedures." Incubations were terminated by extraction with 2 ml of chloroform/methanol (1/1) containing 330 pmol of [2H8]arachidonic acid internal standard. Extracts were analyzed by RP-HPLC to isolate arachidonic acid, which was converted to a pentafluorobenzyl ester derivative and analyzed by GC/MS in negative ion electron capture mode. Selected monitoring of carboxylate anions of arachidonate (m/z 303) and [2H8]arachidonate (m/z 311) was performed to quantitate arachidonate by reference to a standard curve. The amount of arachidonic acid was expressed as a ratio to the lipid phosphorus content of the extract.

Several of our findings suggest a role for iPLA₂ in INS-1 cell signaling responses to cAMP-elevating agents. The group IVA PLA₂ (cPLA₂) has well established signaling functions (5), and, upon cellular activation, cPLA₂ associates with nuclear membranes (79-81). To determine whether iPLA₂ might undergo nuclear association during signaling, we examined the subcellular location of the enzyme in iPLA₂-X INS-1 cells in immunocytofluorescence studies with an antibody generated (71) against peptides in the iPLA₂ sequence. This antibody recognizes an 84-kDa protein corresponding to full-length iPLA₂ in iPLA₂-X INS-1 cells (Fig. 1), and recognition is blocked by including the immunizing peptides in the incubation with the antibody.

Fig. 12 represents an immunocytofluorescence experiment with iPLA₂-X INS-1 cells that were incubated, fixed, permeabilized, treated with iPLA₂ antibody or preimmune antibody, treated with fluorophore-coupled secondary antibody, and examined by confocal fluorescence microscopy. Cells incubated with preimmune antibody yield little fluorescence (upper left), and inclusion of immunizing peptides with iPLA₂ antibody also results in little signal (upper right). iPLA₂-X INS-1 cells incubated at basal glucose without other stimuli exhibit diffuse cytofluorescence and some faint perinuclear halos (lower left). Treating iPLA₂-X INS-1 cells with the cAMP phosphodiesterase inhibitor IBMX induces intense perinuclear fluorescence (lower right), suggesting that iPLA₂ associates with nuclei.

View larger version (62K): [in this window] [in a new window]   Fig. 12.   Immunocytofluorescence study of iPLA2 subcellular distribution in iPLA2-overexpressing INS-1 cells. iPLA2-overexpressing INS-1 cells were allowed to attach to glass slides and incubated in medium that contained 2 mM glucose with 0.1 mM IBMX (lower right panel) or without IBMX (all other panels). Cells were then fixed, permeabilized, and incubated with pre-immune immunoglobulin (upper left) or with iPLA2 antibody (all other panels) with (upper right) or without (all other panels) iPLA2 peptides that had been used to generate the antibody. Secondary antibody coupled to the fluorophore Cy3 was added, and slides were examined by confocal immunofluorescence microscopy as described under "Experimental Procedures." pAb, polyclonal antibody.

A consistent finding was that iPLA₂ immunocytofluorescence in IBMX-treated cells was more intense than that observed for untreated cells (Fig. 12). Similar observations have been reported for immunocytofluorescence studies with cPLA₂ and with the arachidonate 5-lipoxygenase (79), both of which undergo nuclear association in cells treated with calcium ionophores. Two potential explanations of this phenomenon have been proposed (79). One is that there is a greater loss of soluble than of membrane-associated enzyme during the permeabilization and immunocytofluorescence experiment, and the second is that binding to membranes results in better exposure of the epitope recognized by the antibody. The increase in cytoplasmic fluorescence in such cases has been proposed to reflect association with a cytoplasmic membrane structure, such as the endoplasmic reticulum, that is induced by the stimulus for membrane association (79), and similar considerations may apply to iPLA₂.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In the period since the group VIA PLA₂ (iPLA₂) was cloned in 1997 (8-10), a variety of potential functions for the enzyme have been proposed (17, 18, 22-54), based in large part on effects of inhibiting iPLA₂ activity with the suicide substrate BEL. Although BEL inhibits iPLA₂ at concentrations that do not inhibit activities of Ca²⁺-dependent PLA₂ enzymes of the sPLA₂ or cPLA₂ families (4, 11-14), BEL also inhibits PAPH-1 (55) and the recently cloned iPLA₂ (15). Because pharmacologic studies are ambiguous, manipulating iPLA₂ expression by molecular biologic means is an attractive alternative to studying iPLA₂ function. Although suppression of iPLA₂ expression has been achieved with antisense oligonucleotides in murine P388D1 cells (18), these cells express very low levels of iPLA₂, and antisense approaches have not been effective in cells that express higher levels of the enzyme, such as insulin-secreting -cells (47). We have therefore prepared insulinoma cell lines that overexpress iPLA₂ after stable transfection with retroviral constructs containing the iPLA₂ cDNA to study functions of the enzyme.

Two such iPLA₂-overexpressing cell lines exhibit more robust insulin secretory responses to glucose and cAMP-elevating agents than do parental INS-1 cells or cells transfected with empty retroviral vector that does not contain the iPLA₂ cDNA. This suggests that iPLA₂ participates in -cell signaling events involved in insulin secretion, and this is consistent with previous observations (47-54, 82, 83). Synergy between glucose and cAMP-elevating agents in inducing insulin secretion is also a property of native -cells isolated from dispersed islet cells by fluorescence-activated cell sorting, and such cells secrete little insulin in response to glucose unless co-stimulated with agents that increase -cell cAMP (72). This has been taken to indicate that cAMP is required to support glucose-induced insulin secretion from -cells and that paracrine effects of islet -cell-derived glucagon to maintain -cell cAMP levels in intact islets are required for glucose-induced insulin secretion in vivo (72). Agents that elevate cAMP are also known to promote glucose-induced insulin secretion from INS-1 cells and other insulinoma cell lines (73, 74).

Increasing -cell cAMP also represents the mechanism whereby the neuropeptide pituitary adenylase cyclase activating polypeptide and the gut-derived incretins glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide amplify insulin secretory responses to glucose (67, 84-88), and prolactin-induced increases in -cell cAMP participate in up-regulating insulin secretion during islet adaptation to pregnancy (89). In contrast, decreases in -cell cAMP participate in suppression of insulin secretion by epinephrine, somatostatin, prostaglandin E₂, leptin, and neuropeptide Y (66, 90, 91). The mechanisms whereby cAMP augments insulin secretory responses to glucose include an increase in cytosolic [Ca²⁺] resulting from Ca²⁺ entry and mobilization of intracellular Ca²⁺ stores (84, 92-94), and cAMP also sensitizes the exocytotic apparatus to Ca²⁺ (93). Activation of Ca²⁺/calmodulin-dependent cyclic nucleotide phosphodiesterases that degrade cAMP is involved in a feedback loop that limits glucose-induced insulin secretion, and phosphodiesterase inhibitors amplify glucose-induced insulin secretion (95).

It has been reported recently that cAMP restrains -cell PLA₂ activation and arachidonate release and that this limits potentiation of insulin secretion by cAMP-elevating agents in isolated islets (96). Overexpression of iPLA₂ might over-ride this feedback loop and thereby amplify insulin secretion, although the identity of the lipid signal generated by iPLA₂ action is not clearly established. Secretagogue-induced increases in nonesterified arachidonate content are less robust in INS-1 cells than in intact pancreatic islets (47), and lysophospholipid products have been proposed to mediate effects of iPLA₂ activation in some cells (38, 42). The effect of iPLA₂ overexpression on INS-1 cell secretion involves sensitization of the exocytotic apparatus to cAMP rather than augmentation of cAMP accumulation because iPLA₂-overexpressing INS-1 cells do not exhibit higher cAMP levels than control cells when treated with an adenylyl cyclase activator or phosphodiesterase inhibitor, even though iPLA₂-overexpressing cells exhibit more robust insulin secretory responses to such agents in the presence of glucose.

One effect of cAMP-elevating agents in INS-1 cells appears to be to increase nuclear association of iPLA₂. Association of the group IVA PLA₂ (cPLA₂) with nuclei and endoplasmic reticulum (ER) also occurs upon cellular stimulation with agents that induce cPLA₂ activation (79-81), and a similar subcellular distribution is observed for other enzymes involved in arachidonate metabolism in stimulated cells (81, 97). The iPLA₂ deduced amino acid sequence contains a bipartite nuclear localization sequence (45) (⁵¹¹KREFGEHTKMTDVKKPK⁵²⁷) similar to that in nucleoplasmin and some other nuclear proteins in which two adjacent basic amino acids are followed by a flexible spacer region that precedes a second cluster that contains three basic residues (98, 99). Although this sequence might be expected to promote entry into the nucleus and images in Fig. 12 suggest a perinuclear location of iPLA₂, ultrastructural studies are required to determine whether iPLA₂ resides on the cytoplasmic or nucleoplasmic face of the nuclear membrane. The ankyrin repeat domain of iPLA₂ could promote association with either face of the membrane.

Nuclear association of iPLA₂ induced by cAMP-elevating agents in INS-1 cells is of interest because glucose promotes both -cell insulin secretion and proliferation, and glucose-induced INS-1 cell mitogenesis is cAMP-dependent (100). Because membranes of the nucleus and ER are contiguous (79, 94), perinuclear accumulation of iPLA₂ is consistent with association with a subcellular compartment that is likely to include ER (94), and products of PLA₂ action induce Ca²⁺ release from -cell ER (101), which is thought to participate in induction of insulin secretion (102).

Signaling roles for iPLA₂ in other endocrine (22) and secretory (25, 29, 42, 43) events have also been proposed. It is of interest that iPLA₂ is the vastly predominant cytosolic PLA₂ activity in brain (24, 34, 35) and that both brain and islets contain electrically active secretory cells that express many common gene products that are not expressed at comparable abundance in other tissues (103). Among other roles that have been proposed for iPLA₂ are generating substrate for eicosanoid synthesis (26, 30, 32, 33), membrane degradation during apoptosis (27, 39), and regulating membrane phosphatidylcholine composition and content (17, 18, 36, 37). The facts that multiple iPLA₂ splice variants exist (23, 31, 44, 45) that form hetero-oligomers with distinct properties (23) and that iPLA₂ is subject to proteolytic processing that alters its activity (39) suggest that products of the iPLA₂ gene might play multiple roles in cell biology that are dependent on the context of cell-type and maturation or stimulation condition.

One of the earliest proposed roles for iPLA₂ is participation in phospholipid remodeling by generating LPC acceptors for arachidonic acid incorporation into phosphatidylcholine (4, 17, 18). This proposal is based on observations in murine P388D1 cells that inhibiting iPLA₂ activity with BEL (17) or an antisense oligonucleotide (18) suppressed [³H]arachidonic acid incorporation into PC and reduced [³H]LPC levels in [³H]choline-labeled cells. This potential housekeeping function of iPLA₂ is of interest in islets because -cells exhibit the highest levels of arachidonate-containing phospholipids of any known tissue and because such molecules might participate in insulin secretion (69, 82, 83). Previous studies in islets and insulinoma cells indicated that inhibiting iPLA₂ did not suppress arachidonic acid incorporation into -cell PC and reduced LPC levels only slightly (47), and similar findings were obtained with human U937 monocytic cells (77). We re-examined this issue here because it was considered possible that a phospholipid remodeling role of iPLA₂ might be more apparent in cells that overexpress the enzyme. Our ESI/MS measurements indicate, however, that neither iPLA₂ overexpression nor inhibition of iPLA₂ activity affect the rate or extent of incorporation of arachidonic acid into INS-1 cell PC. These and earlier findings (47, 77) argue against a general housekeeping role for iPLA₂ in arachidonate incorporation into phosphatidylcholine.

Our findings are thus more consistent with a signaling rather than a housekeeping role for iPLA₂ in insulin-secreting -cells. The observation that overexpressing iPLA₂ in -cells amplifies their insulin secretory responses could prove to be useful in -cell engineering. Recently, use of modified immunosuppressive regimens has permitted successful transplantation of human islets in seven consecutive patients with insulin-dependent diabetes mellitus, and each patient remained normoglycemic a year after transplantation without exogenous insulin (105, 106). Widespread application of this therapy is precluded by limited availability of donor organs, and -cell lines that are engineered to exhibit regulated insulin secretion could represent an alternate source of cells for transplantation (73, 74, 106). -Cells with improved secretory responses and other properties have been engineered by introducing genes in viral vectors and by clonal selection strategies (104, 107-110). Identifying additional genes whose products affect -cell secretion might permit further progress, and our findings suggest that iPLA₂ gene products might be useful in constructing engineered -cell lines.

	ACKNOWLEDGEMENTS

We thank Karen Green and Dr. Jeffrey Saffitz of the Washington University Department of Pathology for assistance with immunocytofluorescence studies, Dr. Matthew Baker of Research Genetics, Inc. for assistance in peptide antigen selection and generation of the iPLA₂ antibody, Dr. Christopher Newgard (University of Texas Southwestern Medical Center, Dallas, TX) for providing the parental INS-1 cell line, and Denise Kampwerth for assistance in preparing the manuscript.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants R37-DK-34388, P41-RR00954, PO1-HL57278, P60-DK20579, and P30-DK56341; by Career Development Award 2-1999-55 (to Z. M.) from the Juvenile Diabetes Foundation; and by a grant from the American Diabetes Association.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Box 8127, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-8190; Fax: 314-362-8188; E-mail: jturk@imgate.wustl.edu.

Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M010423200

	ABBREVIATIONS

The abbreviations used are: PLA₂, phospholipase A₂; BEL, bromoenol lactone suicide substrate; BSA, bovine serum albumin; cPLA₂, group IV phospholipase A₂; ER, endoplasmic reticulum; ESI, electrospray ionization; GC, gas chromatography; GPC, glycerophosphocholine; IBMX, isobutylmethylxanthine; iPLA₂, group VIA phospholipase A₂; iPLA₂, group VIB phospholipase A₂, MS, mass spectrometry; NP-HPLC, normal phase high performance liquid chromatography; PAPH, phosphatidate phosphohydrolase; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PC, phosphatidylcholine; RP-HPLC, reverse phase high performance liquid chromatography; sPLA₂, secretory phospholipase A₂; TLC, thin layer chromatography; TBS-T, Tris-buffered saline with Tween 20; LPC, lysophosphatidylcholine.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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