β-Cell Calcium-Independent Group VIA Phospholipase A₂ (iPLA₂β)

Abstract

Evidence that group VIA cytosolic calcium-independent phospholipase A₂ (iPLA₂β) participates in β-cell signal transduction includes the observations that inhibition of iPLA₂β with the bromoenol lactone suicide substrate suppresses glucose-stimulated insulin secretion and that overexpression of iPLA₂β amplifies insulin secretory responses in INS-1 insulinoma cells. Immunofluorescence analyses also reveal that iPLA₂β accumulates in the perinuclear region of INS-1 cells stimulated with glucose and forskolin. To characterize this phenomenon further, iPLA₂β was expressed as a fusion protein with enhanced green fluorescent protein (EGFP) in INS-1 cells so that movements of iPLA₂β are reflected by changes in the subcellular distribution of green fluorescence. Stimulation of INS-1 cells overexpressing iPLA₂β-EGFP induced greater insulin secretion and punctate accumulation of iPLA₂β-EGFP fluorescence in the perinuclear region. To determine the identity of organelles with which iPLA₂β might associate, colocalization of green fluorescence with fluorophores associated with specific trackers targeted to different subcellular organelles was examined. Such analyses reveal association of iPLA₂β-EGFP fluorescence with the ER and Golgi compartments. Arachidonate-containing plasmenylethanolamine phospholipid species are abundant in β-cell endoplasmic reticulum (ER) and are excellent substrates for iPLA₂β. Arachidonic acid produced by iPLA₂β-catalyzed hydrolysis of their substrates induces release of Ca²⁺ from ER stores—an event thought to participate in glucose-stimulated insulin secretion.

Phospholipase A₂ (PLA₂) is a diverse group of enzymes that catalyze hydrolysis of the sn-2 substituent from glycerophospholipid substrates to yield a free fatty acid and a 2-lysophospholipid (1,2). Among the PLA₂s is an 84-kDa cytosolic PLA₂ that does not require Ca²⁺ for catalysis and is designated group VIA cytosolic calcium-independent phospholipase A₂ (iPLA₂β) (3–5).

A role for iPLA₂β in signal transduction in insulin-secreting β-cells is suggested by the observations that inhibition of iPLA₂β activity with the bromoenol lactone (BEL) suicide substrate of iPLA₂β suppresses insulin secretion, and overexpression of iPLA₂β amplifies glucose- and forskolin-stimulated insulin secretion from pancreatic islets and INS-1 insulinoma cells (6,7). Stimulation of iPLA₂β-overexpressing INS-1 cells with cAMP-elevating agents also induces translocation of iPLA₂β to the perinuclear region (7). This is of interest because glucose promotes both β-cell insulin secretion and proliferation, and glucose-induced INS-1 cell mitogenesis is cAMP dependent (8). To characterize iPLA₂β subcellular movements further, we developed INS-1 cell lines that overexpress iPLA₂β as a fusion protein with enhanced green fluorescent protein (EGFP) so that green fluorescence associated with EGFP reflects the location of iPLA₂β. Simultaneous monitoring of florescence associated with various tracking molecules allowed us to identify specific subcellular organelles with which iPLA₂β associates when cells are stimulated.

RESEARCH DESIGN AND METHODS

Preparation of a construct for expressing iPLA₂β as a fusion protein with EGFP and selection of stably transfected clones

Full-length iPLA₂β cDNA was amplified by PCR using the following primer set: sense, 5′ AGCTTCGAATTCATGCAGTTCTTTGGACGC-3, and antisense, 5′-TTCGATATCGGGAGATAGCAGCAGCTGG-3′. The amplified full-length iPLA₂β from the pMSCV-neo-iPLA₂β constructs were then subcloned into the pEGFP-N2 (Clontech, Palo Alto, CA) after the immediate early promoter of cytomegalovirus major and before EGFP coding sequences in the same code-reading frame with EGFP. The EGFP-N2 control vector and the construct encoding iPLA₂β-EGFP (FPN2) fusion protein were transfected into INS-1 cells with a Gene PORTER transfection system according to the manufacture’s instructions (Gene Therapy Systems, San Diego, CA). Stably transfected clones were selected using G418 (0.4 mg/ml). Stably transfected cells obtained with EGFP-N2 control vector and FPN2 construct are designed as N2 cells and FPN2 cells, respectively.

Preparation of INS-1 cell subcellular fractions and iPLA₂β enzymatic activity assay

INS-1 cells were washed with PBS and then detached by trituration. Subcellular fractions were prepared, and iPLA₂β activity in aliquots of the fractions (25 µg protein) was assayed and quantitated, as previously described (6).

Immunoblotting analyses of iPLA₂β protein

Western blot analyses were performed with INS-1 subcellular fractions, as previously described (6). The fusion protein was visualized by enhanced chemiluminescence after incubation with primary antibody, anti–green fluorescent protein (GFP) (IgG2α, 0.0002 µg/µl), or anti-iPLA₂β (0.0015 µg/µl) and then the appropriate secondary antibody.

Insulin secretion

Cells were seeded in 24-well plates and allowed to proliferate to confluency. Insulin secretion in response to glucose (0–10 mmol/l) without or with forskolin (2.5 µmol/l) was determined, as described previously (9).

Subcellular organelle tracking

To identify subcellular organelles with which iPLA₂β protein might be associated, EGFP fluorescence (fluorescein isothiocyanate [FITC], green) and fluorescence associated with various organelle markers were monitored in the FPN2-overexpressing cells, as described below. Golgi Tracker (BODIPY TR ceramide) was prepared according to the manufacturer’s instructions (Molecular Probes, Eugene, OR). Cells were first incubated (4°C) with 5 µmol/l ceramide-BSA for 30 min and then washed and incubated (37°C, 1 h) in culture medium containing no glucose and 0.1% BSA. The cells were then incubated (37°C) in medium supplemented with 2 mmol/l glucose and 2.5 µmol/l forskolin for up to 1 h. After various intervals, fluorescent signal from EGFP (FITC) and Golgi Tracker (rhodamine) were monitored.

For tracking of ER, mitochondria, or plasma membrane, cells were first washed and then incubated (37°C, 1 h) in medium containing no glucose and 0.1% BSA. That medium was then replaced with medium containing 2 mmol/l glucose and 2.5 µmol/l forskolin for up to 1 h. The ER tracker (Blue-White DPX, 600 nmol/l) or mitochondrial tracker (Deep Red 633, 100 nmol/l) was added during the final 30 min of incubation. The plasma membrane tracker (DiI D-282, 0.50 µmol/l) was added during the final 20 min of incubation. After various intervals, signals from these fluorophores were monitored.

RESULTS

Immunoblotting analyses of expression of the EGFP fusion protein in INS-1 cells

To demonstrate that transfection of INS-1 cells with the vector containing the iPLA₂β-EGFP construct causes overexpression of the fusion protein, cytosolic and membrane fractions were prepared from transfected INS-1 cells. Proteins in these fractions were analyzed by SDS-PAGE and transferred onto polyvinylidene difluoride membranes that were then used in immunoblotting analyses with antibodies directed against EGFP (Fig. 1A) or iPLA₂β (Fig. 1B). Subcellular fractions prepared from cells transfected with the EGFP vector (N2) alone express only the EGFP-immunoreactive bands of 26 kDa (Fig. 1A, lanes 2 and 4). Transfection with the iPLA₂β-EGFP fusion protein vector (FPN2) causes expression in both the cytosol and membrane fractions of an EGFP-immunoreactive protein with a molecular weight of 110–115 kDa (Fig. 1A, lanes 1 and 3) that represents full-length iPLA₂β as a fusion protein. A second EGFP-immunoreactive band with a molecular weight of 90–95 kDa is also observed in cytosol of FPN2 transfected cells (Fig. 1A, lane 1). This molecular weight corresponds to an iPLA₂β product of β65–70 kDa plus the 26-kDa EGFP. Immunoblotting analyses with iPLA₂β antibody (Fig. 1B) yield results similar to those observed with the GFP antibody. An iPLA₂β-immunoreactive protein corresponding to an iPLA₂β-EGFP fusion protein is not observed in cells transfected with vector containing only the N2 construct (Fig. 1B, lanes 2 and 4), but such a 110- to 115-kDa protein is observed in cells transfected with the vector containing the iPLA₂β-EGFP construct (Fig. 1B, lanes 1 and 3).

FIG. 1.

Immunoblotting analyses of expression of the EGFP- iPLA₂β fusion protein in INS-1 cells. Cytosol and membrane protein fractions were prepared, analyzed by SDS-PAGE, and transferred to polyvinylidene difluoride membranes. The membranes were probed with antibodies directed against GFP (A) or iPLA₂β (B), and the immunoreactive protein bands were visualized by enhanced chemiluminescence. Lanes 1 and 3: FPN2 cells; lanes 2 and 4: N2 cells.

iPLA₂β enzymatic activity in INS-1 cells overexpressing iPLA₂β as a fusion protein

To demonstrate that the FPN2-transfected cells express iPLA₂β enzymatic activity, cytosolic and membrane-associated iPLA₂β activities were assayed in the absence and presence of ATP or BEL. FPN2-transfected cells were found to express calcium-independent PLA₂ activity in cytosol and membranes nearly four- to fivefold higher than that expressed in N2 cells (data not shown). The calcium-independent PLA₂ activity in FPN2 cells is stimulated by ATP and inhibited by the BEL suicide substrate, as is the case for native iPLA₂β (3). These findings and those in Fig. 1 demonstrate that transfection of INS-1 cells with the iPLA₂β-EGFP fusion protein vector causes overexpression of an enzymatically active iPLA₂β protein.

Amplification of glucose-induced insulin secretion by forskolin

INS-1 cells stably overexpressing iPLA₂β exhibit greater insulin secretory responses to cAMP-elevating agents in the presence of glucose (3,6). Similarly, when INS-1 cells that overexpress iPLA₂β as a fusion protein with EGFP are stimulated with the adenylate cyclase activator forskolin, insulin secretary responses are about threefold higher than those of control cells (data not shown).

Secretagogue-stimulated subcellular redistribution of iPLA₂β

Movements of the iPLA₂β-EGFP were monitored in INS-1 cells transfected with FPN2 vectors after secretagogue stimulation for 0.5–2 h. Under basal conditions, the EGFP fluorescence is dispersed. After addition of glucose and forskolin, punctuate accumulations of green fluorescence appear as a halo around the perinuclear region in the FPN2 cells illustrated in the companion article (10). These findings are analogous to our previous observations where antibodies directed against iPLA₂β were used to visualize localization of iPLA₂β (7).

Secretagogue-stimulated redistribution of iPLA₂β to specific organelles

To determine whether iPLA₂β associates with specific organelles in the cell after stimulation, dual fluorescence analyses using organelle trackers were performed. The targeted organelles were the Golgi apparatus, ER, mitochondria, and plasma membrane. FPN2 cells were treated with glucose and forskolin and then loaded with various organelle-specific trackers. The EGFP (green) and organelle tracker fluorescence signals were then recorded in a field of cells. The individual tracker and EGFP fluorescence signals recorded in FPN2 cells are shown in Fig. 2A and B, respectively, and the overlay of the two recordings is shown in Fig. 2C. As illustrated, green fluorescence associated with the iPLA₂β-EGFP protein appears yellow after overlay with the Golgi-rhodamine fluorescence and as blue-white after overlay with the ER-DAPI (4′,6-diamidino-2-phenylindole dihydrochloride) fluorescence. These color changes occur when the two fluorophores colocalize, but not otherwise. There is no apparent overlap of the iPLA₂β-EGFP green fluorescence with the red fluorescence associated with either the mitochondrial or plasma membrane tracker, and the two fluorescent signals remain separate in the overlay image.

FIG. 2.

Secretagogue-stimulated subcellular redistribution of iPLA₂β-EGFP (FPN2). The iPLA₂β-EGFP overexpressing cells (FPN2) were incubated with glucose (2 mmol/l) plus forskolin (2.5 µmol/l) with organelle trackers for Golgi (5 µmol/l), ER (600 nmol/l), mitochondria (100 nmol/l), or plasma membrane (0.50 µmol/l). One hour after stimulation, the EGFP (FITC)-green fluorescent signal was monitored separately from rhodamine-red (Golgi and mitochondrial) and DAPI (4′,6-diamidino-2-phenylindole dihydrochloride)-blue (ER) tracker fluorescent signals. A: Organelle tracker fluorescence alone. B: EGFP fluorescence alone. C: Overlay of EGFP and organelle marker fluorescent signals.

DISCUSSION

Many potential biological functions have been proposed for the cytosolic calcium-independent iPLA₂β enzyme (3). Among the proposed functions are its participation in phospholipid remodeling in P388D1 macrophage–like cells (5,11), cell proliferation (12,13), apoptosis (3), and signal transduction in β-cells and in other cells (12).

In β-cells, inhibition of iPLA₂β suppresses glucose-induced insulin secretion, and overexpression of the enzyme enhances secretion, but neither inhibition nor overexpression of iPLA₂β affects incorporation of fatty acids into β-cell phospholipids (12). These findings indicate that iPLA₂β has a signaling role in insulin secretion but does not serve a housekeeping role in phospholipid remodeling in β-cells.

Immunofluorescence analyses using polyclonal antibodies directed against iPLA₂β also indicate that stimulation of β-cells with insulin secretagogues cause redistribution of iPLA₂β within the β-cell. There is accumulation of an iPLA₂β immunofluorescence signal in the perinuclear region of INS-1 insulinoma cells that are stimulated with glucose and the cAMP-elevating agent forskolin (7). To examine this phenomenon further by an approach not confounded by the potential cross-reactivity of iPLA₂β antibodies, iPLA₂β was overexpressed in INS-1 cells as a fusion protein with EGFP in the studies described here. The location of the fusion protein is reflected by green fluorescence associated with EGFP and facilitates tracking of the subcellular location of iPLA₂β.

In INS-1 cells that overexpress iPLA₂β-EGFP, stimulation with glucose alone or in combination with forskolin induces the appearance of punctate accumulations of green fluorescence as a halo around the perinuclear region. Studies to identify subcellular organelles with which iPLA₂β associates were performed with fluorescent trackers targeted to specific organelles, and these experiments reveal colocalization of iPLA₂β-EGFP–associated green fluorescence with ER and Golgi markers.

Because membranes of the nucleus and ER are contiguous (14,15), perinuclear accumulation of iPLA₂β is consistent with association with a subcellular compartment that is likely to include ER (15). Arachidonate-containing plasmenylethanolamine phospholipid molecular species are abundant in β-cell ER and are excellent substrates for iPLA₂β (16). Arachidonic acid and lysophospholipids produced by iPLA₂β-catalyzed hydrolysis of these substrates induce Ca²⁺ release from β-cell ER, which is thought to participate in induction of insulin secretion (16,17). Arachidonic acid hydrolyzed from plasmenylethanolamine molecular species (18) and/or arachidonate metabolites have been proposed to participate in the β-cell secretory process (17,18), and stimulated association of iPLA₂β with a perinuclear membrane would cause release of arachidonic acid in close proximity to enzymes that catalyze eicosanoid generation and are located in the nuclear envelope and ER (19,20).

Glossary

BEL

bromoenol lactone

EGFP

enhanced green fluorescent protein

FITC

fluorescein isothiocyanate

iPLA₂β

group VIA cytosolic calcium-independent phospholipase A₂

PLA₂

phospholipase A₂