β-Cell Calcium-Independent Group VIA Phospholipase A₂ (iPLA₂β): Tracking iPLA₂β Movements in Response to Stimulation With Insulin Secretagogues in INS-1 Cells

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.

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).

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.

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).

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.

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 ∼65–70 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).

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.

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).

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).

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.

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).

This research was supported by grants from the National Institutes of Health (R37-DK34388, P41-RR00954, P01-HL57278, P60-DK20579, and P30-DK56341) and by an Award (to S.R.) from the American Diabetes Association.

The authors thank Alan Bohrer and Dr. Mary Wohltmann for expert technical assistance.