Apoptosis of Insulin-Secreting Cells Induced by Endoplasmic Reticulum Stress Is Amplified by Overexpression of Group VIA Calcium-Independent Phospholipase A₂ (iPLA₂β) and Suppressed by Inhibition of iPLA₂β

Abstract

The death of insulin-secreting β-cells that causes type I diabetes mellitus (DM) occurs in part by apoptosis, and apoptosis also contributes to progressive β-cell dysfunction in type II DM. Recent reports indicate that ER stress-induced apoptosis contributes to β-cell loss in diabetes. Agents that deplete ER calcium levels induce β-cell apoptosis by a process that is independent of increases in [Ca²⁺]_i. Here we report that the SERCA inhibitor thapsigargin induces apoptosis in INS-1 insulinoma cells and that this is inhibited by a bromoenol lactone (BEL) inhibitor of group VIA calcium-independent phospholipase A₂ (iPLA₂β). Overexpression of iPLA₂β amplifies thapsigargin-induced apoptosis of INS-1 cells, and this is also suppressed by BEL. The magnitude of thapsigargin-induced INS-1 cell apoptosis correlates with the level of iPLA₂β expression in various cell lines, and apoptosis is associated with stimulation of iPLA₂β activity, perinuclear accumulation of iPLA₂β protein and activity, and caspase-3-catalyzed cleavage of full-length 84 kDa iPLA₂β to a 62 kDa product that associates with nuclei. Thapsigargin also induces ceramide accumulation in INS-1 cells, and this response is amplified in cells that overexpress iPLA₂β. These findings indicate that iPLA₂β participates in ER stress-induced apoptosis, a pathway that promotes β-cell death in diabetes.

Diabetes mellitus (DM)¹ is the most prevalent human endocrine disease, and it results from loss and/or dysfunction of insulin-secreting β-cells in pancreatic islets. Type I DM is caused by autoimmune β-cell destruction (1–3), and type II DM results from a progressive decline of β-cell function in the context of insulin resistance (4, 5). Apoptosis contributes to β-cell loss in both type I DM (6) and type II DM (7–12).

Insulin resistance that occurs in rodent models of obesity and in obese humans is associated with an adaptive increase in β-cell mass (13–17). Autopsy studies indicate that the β-cell mass in obese type II DM patients is smaller than that in obese nondiabetic subjects (17–19). The reduction in β-cell mass that occurs in the ZDF rat model of type II DM and in human type II DM patients appears not to be attributable to a reduced level of β-cell proliferation or neogenesis but to an increased level of β-cell death by apoptosis (20, 21).

It is therefore important to understand the mechanisms underlying β-cell apoptosis if this process is to be prevented or retarded. Several stimuli induce β-cell apoptosis (22) by incompletely understood mechanisms. Three currently recognized apoptotic signaling pathways are the extrinsic death receptor pathway, the intrinsic mitochondrial pathway, and the endoplasmic reticulum (ER) stress pathway (23, 24).

ER stress from the loss of Ca²⁺ stores is induced by the SERCA inhibitor thapsigargin (25), and this agent induces insulinoma cell apoptosis by a pathway that does not require an increase in the cytosolic Ca²⁺ concentration but that does require the generation of arachidonic acid 12-lipoxygenase products (26). Thapsigargin-induced ER stress in isolated pancreatic islets is associated with hydrolysis of arachidonic acid from membrane phospholipids by a calcium-independent mechanism that is suppressed by a bromoenol lactone (BEL) inhibitor of group VIA calcium-independent phospholipase A₂β (iPLA₂β) (27). These observations suggest that iPLA₂β participates in ER stress-induced β-cell apoptosis.

A role for iPLA₂β in apoptosis is also suggested by the finding that induction of apoptosis of human U937 promonocytes by anti-Fas antibody is associated with hydrolysis of arachidonic acid from membrane phospholipids by a mechanism that is inhibited by BEL and that is not catalyzed by sPLA₂ or cPLA₂ (28). U937 cell apoptosis is also associated with cleavage of iPLA₂β by caspase-3 (29), a protease that is central to apoptosis execution. Overexpression of this iPLA₂β cleavage product in kidney cells amplifies both thapsigargin-induced arachidonate release and cell death (29), raising the possibility that the caspase-3 cleavage product of iPLA₂β is involved in apoptosis.

Collectively, these observations (26–29) suggest that iPLA₂β might participate in ER stress-induced β-cell apoptosis, and therefore, the effects of overexpression and of inhibition of iPLA₂β on this process were examined here.

MATERIALS AND METHODS

Materials

INS-1 β-cells were generously provided by C. Newgard (Duke University Medical Center, Durham, NC). Other materials were obtained from the following sources: (16:0/[¹⁴C]-18:2)GPC (PLPC, 55 mCi/mmol), rainbow molecular mass standards and enhanced chemiluminescence (ECL) reagent (Amersham Biosciences, Arlington Heights, IL), ceramide and other lipid standards (Avanti Polar Lipids, Alabaster, AL), sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) supplies and Triton X-100 (Bio-Rad, Hercules, CA), retroviral vector (Clontech, Palo Alto, CA), paraformaldehyde (Electron Microscopy Sciences, Ft. Washington, PA), TLC solvents (Fisher, Pittsburgh, PA), RT-PCR reagents (Invitrogen, Carlsbad, CA), normal goat serum and Cy3-conjugated affinipure goat anti-rabbit IgG (H+L) (Jackson Immuno Research Laboratories, West Grove, PA), pentex fraction V fatty acid-free bovine serum albumin (ICN Biomedicals Inc., Aurora, OH), Slow Fade light antifade kit (Molecular Probes, Eugene, OR), caspase-3 inhibitor IV (C3-I, Oncogene Research Products, Boston, MA), Coomasie reagent (Pierce, Rockford, IL), peroxidase-conjugated goat anti-rabbit IgG antibody and TUNEL and DNA Laddering kits (Roche Diagnostic Corp., Indianapolis, IN), and common reagents and salts (Sigma Chemical Co., St. Louis, MO).

Preparation of and Culture of Stably Transfected INS-1 Cells Overexpressing Islet iPLA₂β

A retroviral system (30, 31) was used to stably transfect INS-1 cells with either empty retroviral vector (V) or a vector construct containing iPLA₂β cDNA, as described previously (32). The INS-1 cells were cultured, as described previously (33), and grown to confluency in Petri dishes or flasks prior to being treated with thapsigargin (1 µM).

Preparation of iPLA₂β as a Fusion Protein with EGFP

EGFP-N2 and EGFP-C2 vectors (34) and constructs encoding EGFP and iPLA₂β cDNA were transfected into INS-1 cells with a Gene PORTER transfection system (Gene Therapy Systems, San Diego, CA). To select stably transfected cells that express EGFP or fusion proteins of iPLA₂β-EGFP (N2) or EGFP-iPLA₂β (C2), the selection agent G418 (0.4 mg/mL) was added to culture medium for 2 weeks. Fluorescence-activated cell sorting was then used to increase the efficiency of the selection.

Treatment of Cells and Assessment of Apoptosis

The INS-1 cells were treated with vehicle (DMSO) alone or with thapsigargin (1 fiM) in the absence and presence of inhibitors for up to 24 h. Induction of INS-1 cell apoptosis was then assessed by TUNEL and DNA laddering analyses, performed according to the manufacturer’s instructions.

Preparation of INS-1 Cell Subcellular Fractions and the iPLA₂β Activity Assay

INS-1 cell cytosol and membrane fractions (35) and nuclear enriched fractions (33) were prepared as described previously. The purity of the nuclear fraction, using organelle specific assays (36, 37), and the presence of iPLA₂β activity (38, 39) in this fraction were verified, as described previously (33). The Ca²⁺-independent PLA₂β activity in aliquots of cellular fractions (30 µg of protein) was assayed in the presence of 1-palmitoyl-2-[¹⁴C]-linoleoyl-sn-glycero-3-phosphocholine (PLPC, 5 µM) as the substrate, and specific enzymatic activity was quantitated, as described previously (35, 40).

Immunoblotting and Immunocytofluorescence Analyses of INS-1 Cell iPLA₂β Protein

INS-1 cell protein was analyzed by SDS–PAGE (7.5%), transferred onto Immobilon-P PVDF membranes, and processed for immunoblotting analyses, and iPLA₂β-immunoreactive protein bands were visualized by enhanced chemiluminescence (ECL), as described previously (33). Cellular localization of iPLA₂β protein was achieved using an immunocytofluorescence staining protocol. Following removal of media, cells seeded in chambered glass slides were washed, fixed, permeabilized, and sequentially incubated with iPLA₂β primary antibodies (0.003 µg/µL) and with fluorescent Cy3-conjugated AffiniPure goat anti-rabbit IgG secondary antibody (1:400, 1 h), as described previously (33). Cellular iPLA₂β immunocytofluorescence was then visualized by fluorescence microscopy at an excitation wavelength of 550 nm and an emission wavelength of 570 nm.

Ceramide Analyses

Phospholipids were extracted from INS-1 cells under acidic conditions [2/2/1.8 (v/v/v) chloroform/ methanol/2% acetic acid mixture] as described previously (41, 42). An aliquot of the chloroform layer was saved for PO₄ quantitation. To the organic layer was added C8-ceramide (C8-CM) internal standard (IS, 500 ng), and the mixture was concentrated to dryness under nitrogen. The sample was then analyzed by silica gel G TLC [65/25/4 (v/v/v) chloroform/methanol/30% ammonium hydroxide mixture]. The ceramide standard-containing region was identified with phosphomolybdate staining and ceramide extracted in the presence of 0.6% LiCl to facilitate formation of Li⁺ adducts, as described previously (43, 44). Samples were concentrated and then reconstituted in a 1/4 chloroform/ methanol mixture containing 10 pmol/µL LiOH.

To measure ceramide content, ESI/MS/MS standard curves were generated from a series of samples containing a fixed amount of C8-CM standard and varied amounts of long chain CM standards. The amounts of individual CM species relative to the C8-CM internal standard were measured by ESI/MS/MS scanning for a constant neutral loss (CNL) of 48, which reflects the elimination of formaldehyde and water from the [M + Li⁺]⁺ ion (45). This loss is characteristic of CM−Li⁺ adducts upon low-energy collisionally activated dissociation (CAD) ESI/MS/MS (41). Sample contents of the various CM species were expressed relative to lipid phosphorus, which was measured as described previously (46).

Statistical Analyses

Data were converted to mean ± SEM values where appropriate, and the Student’s t test was applied to determine significant differences between members of a pair (p < 0.05). Statistical differences between multiple treatment groups and a control group were determined using analysis of variances and the Dunnett post-hoc test.

RESULTS

ER Stress-Induced Apoptosis of Parental INS-1 Cells

To examine the effects of ER stress on INS-1 insulinoma cells, nontransfected parental INS-1 cells were treated with vehicle (DMSO) alone or with thapsigargin, and induction of apoptosis was assessed by TUNEL analyses. Figure 1 illustrates that induction of ER stress with thapsigargin induces a 3-fold increase in the level of apoptosis in parental INS-1 cells. This was attenuated by BEL, an inhibitor of iPLA₂β, suggesting that iPLA₂β might be involved in ER stress-induced apoptosis.

Figure 1.

ER stress-induced apoptosis in parental INS-1 cells and effects of BEL inhibition. Parental INS-1 cells were treated with either vehicle (DMSO) alone (Control) or 1 /µM thapsigargin (T) in the absence and presence of 1, 10, or 25 /µM BEL. After 24 h, the cells were harvested for TUNEL analyses. The data representing the percentage of apoptotic cells, relative to the total number of cells, are illustrated as the mean ± SEM (n = 5). The asterisk denotes that the thapsigargin-treated group is significantly different from the control group (p < 0.01). The pound sign denotes that the BEL-treated group is significantly different from the T and Control groups (p < 0.01). The daggers denote that the BEL-treated group is significantly different from the T group (p < 0.05).

Amplification of ER Stress-Induced Apoptosis in Stably Transfected INS-1 Cells that Overexpress iPLA₂β (OE)

The possible nonspecificity of pharmacologic inhibitors such as BEL (47, 48) caused us to pursue a molecular biologic approach in further examining the role of iPLA₂β in apoptosis by manipulating INS-1 cellular levels of iPLA₂β activity. Antisense approaches are ineffective in reducing INS-1 cell iPLA₂β activity (45), but stable transfection with retroviral vectors containing iPLA₂β cDNA has produced several clonal lines that overexpress iPLA₂β (45). Such INS-1 cell lines overexpressing iPLA₂β (OE) were used to further examine the role of iPLA₂β in ER stress-induced apoptosis.

A greater fraction of OE cells subjected to ER stress are TUNEL positive (Figure 2A, right panel) compared to vehicle-treated control OE cells (Figure 2A, left panel). Similarly, ER stress induces a time-dependent increase in the level of DNA laddering in OE cells (Figure 2B). Thus, both the parental INS-1 cell line (Figure 1) and stably transfected INS-1 cells that overexpress iPLA₂β (Figure 2) undergo apoptosis when subjected to ER stress induced by thapsigargin.

Figure 2.

ER stress-induced apoptosis of iPLA₂β-overexpressing INS-1 cells. Stably transfected INS-1 cells that overexpress iPLA₂β (OE) were treated with vehicle alone (Control) or with thapsigargin (T, 1 /µM). After 24 h, the cells were harvested for TUNEL and DNA laddering analyses to determine the fraction of the cell population undergoing apoptosis. (A) The brightly fluorescent cells, denoted with red arrows, reflect apoptotic cells. (B) The lanes marked 0 and 24 contain DNA from OE cells that were treated with thapsigargin for 0 and 24 h, respectively. Lane S contained the standard kilobase ladder, and lane P contained the standard positive sample of DNA from U937 cells provided by the manufacturer.

Quantitative analyses indicate that a greater fraction of iPLA₂β-overexpressing (OE) INS-1 cells undergo apoptosis when subjected to ER stress, relative to vehicle-treated cells, than is the case for INS-1 cells transfected with an empty retroviral vector (V) that contained no iPLA₂β cDNA (Figure 3A). Chelation of extracellular and/or intracellular Ca²⁺ with EGTA and/or BATPA-AM, respectively, does not affect the fraction of OE cells that become apoptotic after being subjected to ER stress (not shown). However, inhibition of iPLA₂β activity with BEL, while not affecting control levels of apoptosis over a 24 h period, greatly reduces the level of ER stress-induced OE cell apoptosis (Figure 3B). These findings support the possibility that iPLA₂β participates in ER stress-induced β-cell apoptosis.

Figure 3.

Effects of iPLA₂β inhibition on ER stress-induced apoptosis in stably transfected INS-1 cells that overexpress iPLA₂β. (A) Stably transfected INS-1 cells that overexpress iPLA₂β (OE cells) or cells that were transfected with the empty retroviral vector (V cells) that contained no iPLA₂β cDNA were treated with vehicle alone (Con) or with thapsigargin (T, 1 µM). After 24 h, the fraction of apoptotic cells was determined with a TUNEL assay. The asterisk denotes that T-treated V cells are significantly different from the corresponding Con group and the pound sign that T-treated OE cells are significantly different from all groups (p < 0.05). (B) OE cells were cultured with vehicle alone (Con) or with thapsigargin (T, 1 µM) in the absence and presence of BEL (1 µM). The asterisk denotes that the T-treated group is significantly different from the Con groups (p < 0.05) and the pound sign that the T- and BEL-treated group is significantly different from other groups (p < 0.05). The data representing the percentage of apoptotic cells, relative to the total number of cells, are illustrated as the mean ± SEM (n = 19).

Correlation between the iPLA₂β Expression Level and Apoptosis in INS-1 Cell Lines Subjected to ER Stress

The potential involvement of iPLA₂β in ER stress-induced apoptosis was further examined by comparing the fraction of cell populations that develop apoptosis among several INS-1 cell lines expressing varying levels of iPLA₂β activity. Figure 4 illustrates that among five such cell lines studied there is a monotonically increasing relationship between the level of iPLA₂β specific activity and the fraction of the INS-1 cell population that is apoptotic after induction of ER stress with thapsigargin. The level of iPLA₂β-immunoreactive protein as assessed by Western blotting analyses correlates well with the level of iPLA₂β activity (Figure 4, inset).

Figure 4.

Relationship between iPLA₂β activity and ER stress-induced INS-1 cell apoptosis. Five distinct INS-1 cell lines that overexpressed varied levels of iPLA₂β activity were treated with thapsigargin (1 µM). After 24 h, the cells were harvested and processed for iPLA₂β activity and TUNEL analyses. The data representing the specific iPLA₂β enzymatic activity and the percentage of apoptotic cells, relative to the total number of cells, are illustrated as the mean ± SEM (n = 6). The inset shows iPLA₂β immunoblotting analyses in 30 µg protein aliquots of homogenates prepared from the five INS-1 cell lines.

Changes in the Subcellular Distribution of iPLA₂β Activity after Induction of ER Stress

Membrane-associated iPLA₂β specific activity exhibits a time-dependent increase for the first 12 h after induction of ER stress and then declines over the next 12 h (Figure 5A). During the first 6 h after induction of ER stress, iPLA₂β specific activity declines in cytosol (Figure 5A) and there is a parallel rise in iPLA₂β activity associated with the nuclear subcellular fraction (Figure 5B). The iPLA₂β activity associated with nuclei then declines over the next 8 h, while that in cytosol rises to a peak at 16 h and then declines.

Figure 5.

ER stress-induced changes in the subcellular distribution of iPLA₂β activity. (A) Aliquots (containing 30 µg of protein) of cytosol and resuspended membranes prepared from OE cells, as described previously (35), were assayed for iPLA₂β activity at various times following treatment with thapsigargin (1 µM). The data are presented as the mean ± SEM (n = 19). The asterisk denotes that the thapsigargin-treated group is significantly different from the 0 h group (p < 0.05). (B) The nuclear fraction was prepared, as described previously (33), from OE cells treated with thapsigargin for various intervals. The data are presented as the mean ± SEM (n = 19) of the percent change in enzymatic activity following thapsigargin treatment, relative to 0 h values. The asterisk denotes that the thapsigargin-treated group is significantly different from the 0 h group (p < 0.05).

Changes in the Subcellular Distribution of the iPLA₂β-ImmunoreactiVe Protein after Induction of ER Stress

Immunocytofluorescence analyses indicate that under control resting conditions iPLA₂β-immunoreactive protein is dispersed in the cytosol of OE cells, and there is a light perinuclear halo (Figure 6A, left panel). Induction of ER stress with thapsigargin causes a marked increase in iPLA₂β perinuclear fluorescence (Figure 6A, right panel). This is associated with a stronger signal for iPLA₂β-immunoreactive protein bands with apparent SDS–PAGE molecular masses of 84, 70, and 62 kDa in the nuclear fraction of OE cells after induction of ER stress with thapsigargin (Figure 6B). The identity of iPLA₂β-immunoreactive protein on such SDS–PAGE gels as authentic iPLA₂β isoforms has recently been confirmed by mass spectrometric analyses.² The relative intensity of the 62 kDa iPLA₂β-immunoreactive band exhibits the greatest increase of the three major distinguishable bands.

Figure 6.

Changes in the subcellular distribution of iPLA₂β-immunoreactive protein after induction of ER stress in INS-1 cells. (A) Cells seeded in glass-chambered slides were cultured in the presence of a vehicle alone or thapsigargin (T, 1 µM) for 24 h and then processed for immunocytofluorescence analyses. Fixed, permeabilized cells were probed with polyclonal iPLA₂β antibodies followed by visualization of Cy3 fluorescence. (B) Aliquots of the INS-1 cell nuclear protein (50 µg) prepared from OE cells treated with thapsigargin (1 µM) were analyzed by SDS—PAGE (7.5%) and transferred onto Immobilon-P PVDF membranes. The electroblot was probed with iPLA₂β antibodies, and immunoreactive protein bands were visualized by enhanced chemiluminescence (ECL): vehicle-treated controls (C) and thapsigargin-treated cells (T).

Further Characterization of the 62 kDa iPLA₂β-Immunoreactive Protein that Accumulates in INS-1 Cells Subjected to ER Stress

The iPLA₂β deduced amino acid sequence contains a consensus sequence (¹⁸⁰DVTD¹⁸³) for cleavage by caspase-3, and caspase-3 is activated during apoptosis (29). Cleavage of rat iPLA₂β at this site would yield a 62 kDa product that retains the ⁴⁶²GTSTG⁴⁶⁶ serine lipase catalytic center (31). We thus postulated that the 62 kDa iPLA₂β-immunoreactive protein that accumulates in the nuclear fraction of INS-1 cells undergoing apoptosis represents the caspase-3 cleavage product of iPLA₂β (N¹⁸⁴–P⁷⁵²).

The iPLA₂β antibody preparation used in these studies was generated by multiple-antigen core technology (Invitrogen) using two peptides [(1) K²⁵—E⁴¹ and (2) M⁵⁸⁹—S⁵⁰²] (Figure 7A) from the rat iPLA₂β deduced amino acid sequence (31). Inclusion of both peptides in incubations with the iPLA₂β antibody blocks recognition of iPLA₂β by competing for and saturating the antibody binding sites. Either peptide alone fails to prevent recognition of full-length iPLA₂β by the iPLA₂β antibody preparation. Since the postulated 62 kDa iPLA₂β caspase-3 cleavage product N¹⁸⁴—P⁷⁵² lacks the region of sequence recognized by peptide 1, we postulated that peptide 2, but not peptide 1, alone would block recognition of the 62 kDa iPLA₂β-immunoreactive protein by the iPLA₂β antibody preparation (Figure 7A).

Figure 7.

Characterization of the interaction of iPLA₂β-immunoreactive proteins with the iPLA₂β antibody preparation. (A) iPLA₂β protein schema. Illustration of peptide antigen sequences in iPLA₂β against which antibodies were generated and location of the consensus sequence for caspase-3 cleavage. (B) Effects of caspase-3 inhibition on the generation of the 62 kDa iPLA₂β-immunoreactive protein postulated to arise from caspase-3 cleavage. The presence of the iPLA2/3-immunoreactive protein was examined in cytosol prepared from cells in the absence (lanes 1 and 3) or presence (lane 2) of a caspase-3 inhibitor (C3-I). Following SDS–PAGE analyses, the proteins were transferred to membranes for immunoblotting studies with the polyclonal iPLA₂β antibody preparation in the absence (lanes 1 and 2) or presence of antigen peptide 2. (C) Fluorescence microscopy was used to visualize iPLA₂β immunocytofluorescence in INS-1 cells probed with iPLA₂β0 antibodies in the absence and presence of neutralizing peptide antigens: (a) iPLA₂β antibodies alone, (b) iPLA₂β antibodies with peptide 2, (c) iPLA₂β antibodies with peptide 1, and (d) iPLA₂β antibodies with peptides 1 and 2.

Consistent with an origin from caspase-3 action, cellular production of the 62 kDa iPLA₂β-immunoreactive protein (Figure 7B, lane 1) is blocked by a caspase-3 inhibitor (C3-I, Figure 7B, lane 2). Recognition of the 62 kDa iPLA₂β-immunoreactive protein is also blocked by peptide 2 (Figure 7B, lane 3) but unaffected by peptide 1 (not shown). However, neither the production of (Figure 7B, lanes 1 and 2) nor the recognition of (Figure 7B, lanes 1 and 3) the 70 and 84 kDa iPLA₂β-immunoreactive proteins is affected by C3-I and peptide 2, respectively. These observations are consistent with the scheme proposed in Figure 7A in which the 62 kDa iPLA₂β-immunoreactive protein represents the caspase-3 cleavage product (N¹⁸⁴—P⁷⁵²) of iPLA₂β and contains the sequence represented by peptide 2 but not that represented by peptide 1, while the 70 and 84 kDa iPLA₂β-immunoreactive proteins retain the region of sequence represented by peptide 1.

A similar antigen blocking strategy was employed to gain information about the subcellular distribution of the iPLA₂β-immunoreactive protein in immunocytofluorescence studies (Figure 7C). INS-1 cells probed with the iPLA₂β antibody preparation alone exhibit immunocytofluorescence dispersed in cytoplasm with a perinuclear halo (Figure 7C, panel a). Inclusion of both antigen peptides 1 and 2 in the incubations with iPLA₂β antibody markedly reduces both cytoplasmic and perinuclear fluorescence (Figure 7C, panel d). Antigen peptide 2 alone (Figure 7C, panel b) but not antigen peptide 1 alone (Figure 7C, panel c) suppresses visualization of perinuclear immunocytofluorescence. This suggests that the perinuclear iPLA₂β-immunoreactive proteins lack the region of sequence near the full-length iPLA₂β N-terminus, and one such protein might be the 62 kDa caspase-3 cleavage product of iPLA₂β (Figure 7A).

Cytofluorescence Studies of INS-1 Cells that Express iPLA₂β as a Fusion Protein with EGFP

Immunocytofluorescence studies can be confounded by the cross reactivity of the antibody with proteins other than the intended target. Cytofluorescence studies (Figure 8) were therefore performed with INS-1 cells transfected with vectors (panel A) designed to produce EGFP alone (panels B and C), or iPLA₂β as a fusion protein with the fluorescent EGFP tag at the N-terminus (EGFP-iPLA₂β, panels D and E) or at the C-terminus (iPLA₂β-EGFP, panels F and G). INS-1 cells that produced either iPLA₂β-EGFP or EGFP-iPLA2/3 develop apoptosis after induction of ER stress with thapsigargin (not shown).

Figure 8.

Effects of ER stress on the subcellular distribution of an iPLA₂β-EGFP fusion protein in INS-1 cells. (A) Schematic of two fusion protein constructs where X indicates the potential caspase-3 cleavage site in the iPLA₂β protein. INS-1 cells were transfected with either EGFP vector alone (panels B and C) or constructs designed to attach the EGFP tag to the N-terminus (EGFP-iPLA₂β, panels D and E) or the C-terminus (iPLA₂β-EGFP, panels F and G) of iPLA₂β. Cells were treated with vehicle only (Control, panels BD, and F) or with 1 µM thapsigargin (T, panels CE, and G) and examined by fluorescence microscopy 6 h later. The iPLA₂β-EGFP-producing INS-1 cells treated with thapsigargin exhibit punctate perinuclear iPLA₂β fluorescence, as indicated with yellow arrows (panel E).

Upon treatment with the vehicle (DMSO) for 6 h, INS-1 cells that produce EGFP alone (Figure 8, panel B), iPLA₂β-EGFP (panel D), or EGFP-iPLA₂β (panel F) exhibit diffuse fluorescence with little evidence of compartmentalization. After induction of ER stress for 6 h with thapsigargin, INS-1 cells producing EGFP alone continue to exhibit diffuse fluorescence (panel C). The iPLA₂β-EGFP-producing cells, however, exhibit punctuate perinuclear fluorescence (panel E). In contrast, EGFP-iPLA₂β-producing cells exhibited diffuse cytoplasmic fluorescence after induction of ER stress, and there is no evidence of perinuclear localization (panel G). In fact, fluorescence was excluded from the nuclear region.

An interpretation of the findings illustrated in Figure 8 that is consistent with the scheme proposed in Figure 7A and with the discussion of findings in panels B and C of Figure 7 is that it is the C-terminal product of caspase-3 cleavage of iPLA₂β that accumulates in the perinuclear region of INS-1 cells subjected to ER stress. This C-terminal product bears a fluorescent label when the substrate is iPLA₂β-EGFP but not when it is EGFP-iPLA₂β.

To determine whether inhibition of caspase-3 affects the subcellular redistribution of iPLA₂β-immunoreactive proteins induced by ER stress in INS-1 cells, the caspase-3 inhibitor C3-I was used in iPLA₂β immunocytofluorescence studies of thapsigargin-treated cells (Figure 9). Compared to vehicle-treated control cells (Control panel) or to cells treated with C3-I alone (C3-I panel), INS-1 cells subjected to ER stress with thapsigargin in the absence of C3-I (T panel) exhibit intense perinuclear iPLA₂β immunocytofluorescence, and this response is greatly attenuated by C3-I (T + C3-I panel). BEL does not affect the ER stress-induced perinuclear accumulation of iPLA₂β immunoreactivity (not shown) under conditions where C3-I does so.

Figure 9.

Effects of caspase-3 inhibition on the subcellular redistribution of iPLA₂β immunocytofluorescence in INS-1 cells subjected to ER stress. OE cells were pretreated with vehicle alone or with caspase-3 inhibitor (C3-I, 500 nM) for 24 h. The vehicle-pretreated cells were then treated with vehicle alone (Control) or with 1 /µM thapsigargin (T) alone, and the C3-I-pretreated cells were treated with C3-I alone (C3-I) or with T (T + C3-I). After an additional 24 h, the cells were processed for iPLA₂β immunocytofluorescence analyses by fluorescence microscopy. The data are representative of findings obtained from three separate experiments.

Inhibition of caspase-3 also greatly reduces the fraction of the INS-1 cell population that develops apoptosis after induction of ER stress (Figure 10), and the magnitude of this effect is similar to that observed with BEL (Figure 3B). The incidence of apoptosis in INS-1 cells treated with C3-I alone is similar to that exhibited by vehicle-treated cells (Figure 10). Taken together, the findings illustrated in Figures 5–10 suggest that ER stress in INS-1 cells induces the caspase-3-catalyzed cleavage of iPLA₂β to yield a product that retains the native C-terminus, lacks a portion of the native N-terminus, accumulates in the perinuclear region, retains catalytic activity, and plays a functional role in the development of apoptosis.

Figure 10.

Effects of inhibition of caspase-3 on inhibition of ER stress-induced apoptosis of INS-1 cells. OE cells were pretreated with vehicle alone or with 500 nM C3-I for 24 h. The vehicle-pretreated cells were then treated with vehicle alone (Control) or with 1 µM thapsigargin (T) alone, and the C3-I-pretreated cells were treated with either C3-I alone (C3-I) or T (T + C3-I). After 24 h, cells were harvested and TUNEL analyses performed. The data representing the percentage of apoptotic cells, relative to the total number of cells, are illustrated as the mean± (SEM (n =10). The asterisk denotes that the T group is significantly different from Control and C3-I groups and the pound sign that the T + C3-I group is significantly different from other groups (p < 0.05).

ER Stress-Induced Ceramide Accumulation in INS-1 Cells

Ceramide is an important lipid mediator of apoptosis (49–51), and recent reports suggest that iPLA₂β activation promotes ceramide accumulation (52, 53). We therefore examined whether ER stress-induced apoptosis is associated with ceramide accumulation in INS-1 cells and whether iPLA₂β overexpression amplifies this response.

Ceramide (CM) species were measured as their Li⁺ adducts (42) relative to the internal standard C8-CM, which contains an octanoic acid residue as the fatty amide substituent and is not an endogenous component of INS-1 cell lipids. Figure 11A is the positive ion ESI-MS total ion current (TIC) tracing of Li⁺ adducts of the CM species in INS-1 cell lipids after addition of the C8-CM internal standard (IS), which is represented in the spectrum by its [M + Li⁺]⁺ ion (m/z 432). The fatty amide substituents of the major CM species endogenous to INS-1 cells are 24:0 (m/z 656), 24:1 (m/z 654), 22:0 (m/z 628), 18:0 (m/z 572), and 16:0 (m/z 544).

Figure 11.

Electrospray ionization (ESI) mass spectrometric analyses of ceramide species from INS-1 cells as Li⁺ adducts. Ceramides were extracted from INS-1 cells and analyzed by ESI/MS as Li⁺ adducts. (A) Positive ion ESI/MS total ion current (TIC). (B) ESI/MS tandem spectrum obtained from collisionally activated dissociation (CAD) of m/z 654 (C24:1–CM-Li⁺). (C) ESI/MS tandem spectrum obtained from CAD of m/z 544 (C16:0–CM-Li⁺). (D) Rationalization of the ceramide (24:1-CM) fragmentation pattern. R is the fatty acid substituent side chain.

Panels B and C of Figure 11 are the tandem mass spectra produced by collisionally activated dissociation (CAD) of the [M + Li⁺]⁺ ions of C24:1-CM (m/z 654) and C16:0-CM (m/z 544), respectively. Both spectra contain prominent ([M + Li⁺]⁺ − 48) ions at m/z 606 (Figure 11B) and m/z 496 (Figure 11C), respectively, that reflect sequential neutral losses of water and form aldehyde (42). The two spectra contain the common ion at m/z 264 that arises from and identifies the long chain base (42). Ions that arise from and identify the fatty amide substituent (42) in Figure 11B include those at m/z 372 and 398, and analogous ions in Figure 11C occur at m/z 262 and 288. Formation of these ions is rationalized in Figure 11D.

Figure 12 illustrates the application of monitoring a constant neutral loss (CNL) of 48 (42, 45) to the measurement of CM species in INS-1 cells that overexpress iPLA₂β after incubation for 24 h under control conditions (Figure 12A) or after induction of ER stress with thapsigargin (Figure 12B). In comparison to the abundances of CM species in control cells (Figure 12A), an increase in the abundance of all of the major endogenous CM species in the INS-1 cells subjected to ER stress (Figure 12B) is reflected by the increased intensity of ions that represent them (e.g., m/z 544, 572, 628, 654, and 656), relative to that for the C8-CM IS (m/z 432).

Figure 12.

ER stress-induced ceramide accumulation in INS-1 cells. Ceramides extracted from OE cells incubated for 24 h with vehicle alone (A) or with thapsigargin (B) were analyzed as Li⁺ adducts by ESI/MS/MS scanning for a constant neutral loss of 48.

Figure 13 illustrates that ER stress-induced ceramide accumulation is time-dependent over 24 h and that this effect is much larger in stably transfected cells that overexpress iPLA₂β (OE cells) than in INS-1 cells transfected with the empty retroviral vector (V cells). These findings suggest that enhanced ceramide accumulation might contribute to the stronger tendency of OE cells to undergo apoptosis in response to ER stress than V cells (Figure 3).

Figure 13.

Time-course of ER stress-induced ceramide accumulation in INS-1 cells and effects of iPLA₂β overexpression. The ceramide (CM) content was measured by ESI/MS/MS in lipids extracted from INS-1 cells transfected with the empty vector (V cells) or stably transfected INS-1 cells that overexpress iPLA2/3 (OE cells) treated for various periods with thapsigargin (1 µM). The CM content was normalized to the total lipid phosphorus content and expressed as the ratio to the normalized CM content of the time zero value. (A) Change in content with time after exposure to thapsigargin for individual CM molecular species. (B) Time course of accumulation of the sum of all CM species. The asterisks denote that thapsigargin-treated OE-cell groups are significantly different from the corresponding 0 h OE group (p < 0.01). The dagger denotes that the V cell group at 24 h is significantly different from the corresponding 0 h V cell group and OE group at 24 h (p < 0.05). The pound signs denote that the V cell groups at 6 and 16 h are significantly different from the corresponding OE groups (p < 0.05).

DISCUSSION

Apoptosis plays a prominent role in the loss of β-cells during development of type I diabetes (6), and type II diabetes is characterized by an initial peripheral insulin resistance and accompanying compensatory hyperinsulinemia that is followed by a loss in β-cell function and frank hyperglycemia (4, 5). The progressive decrease in β-cell function in type II diabetes reflects, in part, β-cell death by apoptosis (7–12).

Three distinct recognized pathways for apoptosis include the (extrinsic) death receptor, the (intrinsic) mitochondrial, and the ER stress pathways (23, 24, 43, 54, 55). All three pathways result in activation of caspase-3 which is an executioner of apoptosis (24, 44). ER stress-induced apoptosis has been reported to contribute to progressive β-cell death in the Akita diabetic mouse model (56) and to β-cell death induced by nitric oxide and the SERCA inhibitor thapsigargin (57, 58), a known inducer of ER stress (23, 54).

The potential deleterious effects of intracellular calcium store depletion on β-cell survival were previously also examined in MIN-6 insulinoma cells (26). In that study, depletion of ER calcium stores in MIN-6 insulinoma cells by thapsigargin was found to induce apoptosis by a mechanism that does not require an increase in the intracellular calcium concentration, but that does require generation of the arachidonic acid 12-lipoxygenase product 12-HETE (26). This implies that thapsigargin induces β-cell hydrolysis of arachidonic acid from membrane phospholipids by a mechanism that does not require a rise in the cytosolic Ca²⁺ concentration, and this has been demonstrated to occur in pancreatic islets (27) by a pathway that is suppressed by BEL, a suicide substrate inhibitor of the group VIA phospholipase A₂ (iPLA₂β). Similarly, the cytokine IL-1 induces β-cell NO production and apoptosis and hydrolysis of arachidonic acid from membrane phospholipids by a mechanism that is also sensitive to inhibition by BEL (59). These observations suggest that iPLA₂β might participate in membrane phospholipid hydrolysis in β-cells in which apoptosis is induced by exposure to IL-1 or induction of ER stress.

The possibility that iPLA₂β participates in ER stress-induced apoptosis was examined here in iPLA₂β-expressing (32, 33, 45, 46) INS-1 insulinoma cells, and the findings reveal that induction of ER stress in parental INS-1 cells with thapsigargin induces apoptosis, and that BEL suppresses this response in a concentration-dependent manner. This is consistent with a role for iPLA₂β in ER stress-induced β-cell apoptosis. Supporting this role for iPLA₂β is the finding that anti-Fas antibody-induced apoptosis of human promonocytic U937 cells is associated with release of arachidonic acid that is not mediated by cPLA₂ or sPLA₂ but is inhibited by BEL (28).

Our finding that ER stress-induced apoptosis of INS-1 cells is suppressed by BEL caused us to determine whether overexpression of iPLA₂β in INS-1 cells would affect their sensitivity to ER stress. Overexpression of iPLA₂β did amplify the INS-1 cell apoptotic response to thapsigargin, and this response is also suppressed by BEL. In addition, thapsigargin-induced apoptosis of INS-1 cells is associated with stimulation of iPLA₂β activity within 1 h of exposure, and the enzymatic activity continues to increase for 12 h. Moreover, enzymatic, immunoblotting, and immunocytofluorescence analyses all demonstrate a thapsigargin-induced perinuclear accumulation of iPLA₂β protein and enzymatic activity and the accumulation of a 62 kDa iPLA₂β-immunoreactive protein in the nuclear fraction.

Caspase-3-catalyzed cleavage of iPLA₂β at its ¹⁸⁰DVTD¹⁸³ consensus site (29, 44) would yield a 62 kDa product that retains the ⁴⁶²GTSTG⁴⁶⁶ lipase catalytic center and lacks a region of N-terminal sequence that includes one of the two antigenic peptide sites (peptide 1) recognized by our iPLA₂β antibody preparation. Such a 62 kDa product would retain the other antigenic peptide site (peptide 2). Consistent with an origin of the INS-1 cell 62 kDa iPLA₂β-immunoreactive protein from caspase-3 action, peptide 2, but not peptide 1, blocks recognition of this immunoreactive protein by the iPLA₂β antibody. In addition, inhibition of caspase-3 activity reduces the level of perinuclear accumulation of iPLA₂β immunocytofluorescence, generation of 62 kDa iPLA₂β-immunoreactive band, and nuclear accumulation of iPLA₂β enzymatic activity. These findings suggest induction of ER stress with thapsigargin promotes caspase-3-catalyzed cleavage of INS-1 cell iPLA₂β.

The caspase-3 inhibitor also reduces INS-1 cell apoptosis following thapsigargin treatment, consistent with the recognized role of caspase-3 as an executioner of apoptosis (24, 44). Induction of apoptosis in human promonocytic U937 cells is also associated with caspase-3-catalyzed cleavage of iPLA₂β (29), and overexpression of this truncated form (amino acid 184 → C-terminus) of iPLA₂β in human embryonic kidney cells enhances both apoptosis and AA release (29). These findings suggest that the product of caspase-3-catalyzed iPLA₂β is more active than the full-length iPLA₂β, and it has been suggested that the more active shorter iPLA₂β isoform hydrolyzes nuclear membrane phospholipids, which disrupts membrane fluidity and results in apoptosis. Though BEL was effective in suppressing iPLA₂β activity over the study period following thapsigargin treatment, it did not appear to attenuate perinuclear accumulation of the iPLA₂β protein or significantly inhibit generation of a 62 kDa iPLA₂β-immunoreactive product. These observations suggest that BEL is affecting iPLA₂β downstream of caspase-3. Our findings that thapsigargin stimulates iPLA₂β activity and promotes caspase-3-catalyzed cleavage of INS-1 cell iPLA₂β and accumulation of the resultant 62 kDa iPLA₂β-immunoreactive product in the perinuclear region suggest that these events may be involved in the mechanism by which iPLA₂β participates in ER stress-induced β-cell apoptosis.

INS-1 cells that overexpress iPLA₂β also accumulate larger amounts of ceramide when treated with thapsigargin than do control INS-1 cells. Ceramides are lipid messengers that suppress cell growth and induce apoptosis (49–51). They can be generated via sphingomyelin hydrolysis by sphingomyelinase, resulting in the generation of ceramide and phosphocholine (60) or by de noVo synthesis from palmitate (16:0) and other precursors. This latter mechanism is thought to be involved in lipoapoptosis of β-cells (61). Ceramide accumulation can also result from degradation of ceramidase, a pathway that is promoted by nitric oxide (52). Increases in the level of ceramide generation have been linked to arachidonic acid levels in colorectal cancer cells and HL-60 cells (62, 63), and IL-1β-induced arachidonic acid generation from pancreatic islets has been shown to be sensitive to inhibition by BEL (59). Taken together with the recent demonstration that LPS-induced generation of nitric oxide is inhibited by BEL (53), these findings raise the possibility that iPLA₂β activation could enhance NO production and cause degradation of ceramidase, accumulation of ceramide, and amplification of apoptosis.

ER stress-induced β-cell apoptosis is now thought to be a potentially important factor in the development of diabetes (58). As membranes of the nucleus and ER are contiguous (64, 65), perinuclear accumulation of iPLA₂β is consistent with association of the iPLA₂β protein with a subcellular compartment that is likely to include ER (64). The β-cell ER is enriched in arachidonate-containing plasmenyletha-nolamine species (40), and products of PLA₂ action might participate in apoptosis, as has been previously suggested (26, 59, 66–68). Our findings support the possibility that iPLA₂β and the products of its action participate in β-cell apoptosis.