Stimulation of insulin secretion and associated nuclear accumulation of iPLA2beta in INS-1 insulinoma cells

Zhongmin Ma1, Sheng Zhang2, John Turk2, and Sasanka Ramanadham2

1 Division of Experimental Diabetes and Aging, Mount Sinai School of Medicine, New York, New York 10029; and 2 Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Accumulating evidence suggests that the cytosolic calcium-independent phospholipase A2 (iPLA2beta ) manifests a signaling role in insulin-secreting (INS-1) beta -cells. Earlier, we reported that insulin-secretory responses to cAMP-elevating agents are amplified in iPLA2beta -overexpressing INS-1 cells (Ma Z, Ramanadham S, Bohrer A, Wohltmann M, Zhang S, and Turk J. J Biol Chem 276: 13198-13208, 2001). Here, immunofluorescence, immunoaffinity, and enzymatic activity analyses are used to examine distribution of iPLA2beta in stimulated INS-1 cells in greater detail. Overexpression of iPLA2beta in INS-1 cells leads to increased accumulation of iPLA2beta in the nuclear fraction. Increasing glucose concentrations alone results in modest increases in insulin secretion, relative to parental cells, and in nuclear accumulation of the iPLA2beta protein. In contrast, cAMP-elevating agents induce robust increases in insulin secretion and in time-dependent nuclear accumulation of iPLA2beta fluorescence, which is reflected by increases in nuclear iPLA2beta protein content and specific enzymatic activity. The stimulated effects are significantly attenuated in the presence of cell-permeable inhibitors of protein phosphorylation and glycosylation. These findings suggest that conditions that amplify insulin secretion promote translocation of beta -cell iPLA2beta to the nuclei, where it may serve a crucial signaling role.

immunofluorescence; immunoaffinity; enzymatic activity; insulin secretion; nuclear localization


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

PHOSPHOLIPASES A2 (PLA2) are a diverse group of enzymes that catalyze the hydrolysis of the sn-2 substituent from glycerophospholipid substrates to yield a free fatty acid and a 2-lysophospholipid (9, 12). Among the PLA2s is an 85-kDa cytosolic PLA2 that does not require Ca2+ for catalysis. This PLA2 has now been cloned from several sources (3, 23, 44), including rat and human pancreatic islet beta -cells (23, 25), and has been proposed to be designated as the beta -isoform (26, 43) of Group VIA calcium-independent PLA2, or iPLA2beta (22, 26). Several potential functions for iPLA2beta have been proposed, including a housekeeping role (4) in phospholipid remodeling and a signaling role in secretion (22, 23, 29).

Because glucose-stimulated insulin secretion is associated with increased hydrolysis of the sn-2 substituent arachidonate from beta -cell membrane phospholipids and its accumulation within the beta -cells (45, 48), we initially investigated the possible participation of iPLA2beta in the insulin-secretory pathway. Those studies (34, 37), performed in insulinoma cells and native pancreatic islets, revealed that inhibition of iPLA2beta by the bromoenol lactone (BEL) suicide inhibitor of iPLA2beta suppresses both glucose-stimulated arachidonate stimulation and insulin secretion. These findings raise the possibility that iPLA2beta serves a signaling role in beta -cells.

Subsequent findings in studies with murine P388D1 macrophage-like cells have led to the proposal that the iPLA2beta enzyme serves a housekeeping role in phospholipid remodeling that involves generation of lysophospholipid acceptors for incorporation of arachidonic acid into phospholipids (4). However, detailed examination of this process by our group (35) indicated that inhibition of the iPLA2beta enzyme does not influence incorporation of arachidonate or phospholipid remodeling in pancreatic islets or insulinoma cells but does inhibit secretagogue-stimulated insulin secretion.

The potential signaling role of iPLA2beta was further addressed in a recently reported study (22), in which the consequence of overexpressing iPLA2beta on insulin secretion from INS-1 insulinoma cells and of phospholipid remodeling in these cells was examined. INS-1 cells after prolonged passaging become less responsive to glucose-stimulated insulin secretion (36). Late passage cells were transfected with iPLA2beta cDNA, and the iPLA2beta -overexpressing INS-1 cells were subsequently incubated with glucose, either alone or in combination with cAMP-elevating agents. In contrast to the parental INS-1 cells, which were unresponsive to glucose and only mildly responsive to cAMP-elevating agents, a mild improvement in glucose responsiveness but robust responses to IBMX and forskolin were evident in the iPLA2beta -overexpressing INS-1 cells (22). Improvements in the insulin-secretory responses, however, were not associated with alterations in arachidonic acid incorporation into phospholipids of the transfected cells. Collectively, these findings reaffirm a signaling role for iPLA2beta in insulin-secreting beta -cells.

An intriguing finding in the study just described (22) was that stimulation of iPLA2beta -overexpressing INS-1 cells with cAMP-elevating agents was associated with increased accumulation of iPLA2beta in the nuclear region (22). In the present study, this phenomenon is further examined by use of immunocytofluorescence, immunoaffinity, and enzymatic activity analyses, and contributions of possible posttranslational modifications of the enzyme are addressed.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. INS-1 cells were generously provided by Dr. C. Newgard (University of Texas Southwestern Center for Diabetes Research, Dallas, TX). Other materials were obtained from the following sources: {[gamma -32P]ATP, (16:0/[14C]18:2)-glycerophosphocholine [1-palmitoyl-2-[14C]linoleoyl-sn-glycero-3-phosphocholine (PLPC), 55 mCi/mmol]} rainbow molecular mass standards, and enhanced chemiluminescence (ECL) reagent from Amersham (Arlington Heights, IL); H-89 from Biomol Research Laboratories (Plymouth Meeting, PA); SDS-PAGE supplies from Bio-Rad (Richmond, CA); pentex fraction V fatty acid-free BSA from ICN Biochemicals (Aurora, OH); normal goat serum and Cy3-conjugated AffiniPure goat anti-rabbit IgG (H+L) from Jackson Immuno Research Laboratories (West Grove, PA); Enlightning solution, Slow Fade light antifade kit from Molecular Probes (Eugene, OR); [3H]AMP (25 Ci/mmol), UDP [3H]galactose (48 Ci/mmol) from New England Nuclear (Boston, MA); bezyl N-acetyl-alpha -D-galactosaminide (BG), GlcNac Kit from Oxford Glycosystems (Wakefield, MA); Coomassie reagent from Pierce (Rockford, IL); alkaline phosphatase, peroxidase-conjugated goat anti-rabbit IgG antibody from Roche Diagnostic (Indianapolis, IN); bisbenzimide (Hoechst no. 33258), catalytic protein kinase A (cPKA), forskolin (FSK), fraction V bovine albumin, genistein, globulin-free bovine albumin, IBMX, NP-40, protease inhibitor cocktail (PIC), staurosporine, and common reagents and salts from Sigma Chemical (St. Louis, MO); antibiotic solutions and cell culture media from the Tissue Culture Support Center, Washington University (St. Louis, MO); protein kinase A inhibitor (PKAI) from UBI (Lake Placid, NY); and Oct-1 (C-21) antibody from Zymed Laboratories (San Francisco, CA).

Preparation and culture of stably transfected INS-1 cells that overexpress islet iPLA2beta . A retroviral system (6, 23) was used to stably transfect INS-1 cells (at ~passage 70) with iPLA2beta cDNA and achieve overexpression, as described (22). Freshly collected retrovirus-containing medium was added to INS-1 cell monolayers, and stably transfected cells expressing high levels of iPLA2beta were selected by culturing retrovirally infected cells with medium containing G-418 (0.4 mg/ml). The expanded iPLA2beta -overexpressing INS-1 cells were cultured, as described (11), in RPMI 1640 medium, which contained 11 mM glucose, 10% fetal calf serum, 10 mM HEPES buffer, 2 mM glutamine, 1 mM sodium pyruvate, 50 mM beta -mercaptoethanol (BME), and 0.1% (wt/vol) each of penicillin, fungizone, and streptomycin. RetroPack PT 67 cells (Clontech, Palo Alto, CA) were maintained in DMEM (4.5 mg/ml glucose) containing 10% FBS, 4 mM L-glutamine, 100 U/ml of penicillin, and 100 µg/ml of streptomycin.

Preparation of INS-1 cell nuclear fraction. iPLA2beta -overexpressing INS-1 cells, grown to confluence in T75 flasks, were washed with phosphate-buffered saline (PBS) and detached by incubation (3 min, 37°C) with 0.05% trypsin-0.02% EDTA. The cells were transferred to Tris-buffered saline (TBS) and pelleted by centrifugation (1,500 g for 5 min). Nuclear extracts were prepared, as described (42), by resuspending the cells in 400 µl of cold buffer A [10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol (DTT) and 10 µl PIC/106 cells] by gentle trituration. The cells were allowed to swell on ice for 15 min, and then 10 µl of freshly prepared 1% NP-40 (final concentration 0.025%) were added. After vigorous vortexing for 10 s, the cell homogenate (CH) was centrifuged (16,000 g, 30 s, 4°C). The supernatant, containing cytoplasm and RNA, was saved and designated nonnuclear (NN) material. The nuclear pellet was resuspended in 50 µl of ice-cold buffer B (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and PIC) and rocked vigorously (15 min, 4°C). The nuclear homogenate (NH) was centrifuged (16,000 g, 5 min, 4°C), the nuclear extract (NE) supernatant was saved, and the particulate (P) material was resuspended in buffer B (25 µl). Protein content in each fraction was determined by Bio-Rad assay against the BSA standard.

Verification of purity of nuclear preparation. To ensure that the nuclear fraction did not contain mitochondria or plasma membrane fractions that might contribute to measured iPLA2beta activity, cytochrome c oxidase (mitochondrial marker enzyme) and 5'-nucleotidase (plasma membrane marker enzyme) activity assays were performed, as described (33, 42). In addition, aliquots of the cellular fractions were processed for immunoaffinity analyses, as we will describe, for the nuclear marker Oct-1. Incubations with primary (1:1,000) and secondary (1:40,000) antibodies were followed by visualization of immunoreactive bands by ECL.

iPLA2beta enzymatic activity assay. Enzymatic Ca2+-independent PLA2beta activity in aliquots of cellular fractions (30 µg of protein) were assayed by ethanolic injection (5 µl) of the substrate PLPC (5 µM) in assay buffer (40 mM Tris, pH 7.5, and 5 mM EGTA, total volume 200 µl). Assay mixtures were incubated (5 min, 37°C, with shaking), and the assay reaction was terminated with butanol (100 µl) addition and vigorous vortexing. The reaction mixture was centrifuged (2,000 g, 5 min), and products in the upper butanol layer were analyzed by silica gel G thin-layer chromatography (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, and the released 14C fatty acid was quantitated by liquid scintillation spectrometry. Specific iPLA2beta activity was calculated from the dpm of released fatty acid and protein content as described (14). To verify that the measured activity reflected that of the iPLA2beta (35), the abilities of ATP (10 mM) to stimulate and of BEL (10 µM), a suicide inhibitor of iPLA2beta , to inhibit activity were examined. To test the effects of BEL on activity, the sample protein was preincubated with BEL [2 min at room temperature (RT)] before addition of substrate.

Immunoaffinity analyses of INS-1 cell iPLA2beta protein. INS-1 cellular fractions were diluted in an equal volume of sample buffer (1 M Tris · HCl, pH 6.8, 69 mM SDS, 10% glycerol, 0.01% bromophenyl blue, and 1% BME) and boiled. Aliquots containing 25-50 µg of protein were analyzed by SDS-PAGE (7.5%) and transferred onto Immobilon-P polyvinylidene difluoride (PVDF) membrane. The electroblot was sequentially blocked (3 h at RT) with TBS-Tween 20 (TBS-T; 20 mM Tris · HCl, 137 mM NaCl, pH 7.6, and 0.05% Tween-20) containing 5% milk protein, washed (TBS-T, 5 × 5 min), and then incubated (1 h, RT) with purified polyclonal (p) antibodies (0.0015 µg/µl in TBS-T containing 3% BSA) to iPLA2beta generated by multiple antigen core technology (Research Genetics, Huntsville, AL) against peptides in the iPLA2beta -deduced amino acid sequence. The iPLA2beta peptides coupled to this core for immunizing rabbits were 25KEVSLADYASSERVRE41 and 489RMKDEVFRGSRPY502. The membrane was again washed in TBS-T (5 × 5 min) and incubated (1 h, RT) with peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:40,000 in TBS-T containing 3% BSA). The immunoreactive protein bands were visualized by ECL.

Visualization of iPLA2beta protein by immunofluorescence staining and confocal microscopy. After removal of media, the attached cells were sequentially washed with PBS (2 × 5 min), fixed in 4% paraformaldehyde (10 min), washed (3 × 5 min), permeabilized in cold methanol (20 min, -20°C), washed (3 × 5 min), and blocked (in PBS containing 1% globulin-free BSA, 0.30% Triton X-100, and 3% normal goat serum). Next, piPLA2beta primary antibodies (0.003 µg/µl) were added, and the cells were incubated in a humidified chamber (O/N, 4°C). The cells were washed free of the primary antibodies (3 × 5 min) and incubated with fluorescent Cy3-conjugated AffiniPure goat anti-rabbit IgG secondary antibody (1:400, 1 h). The cells were then washed (PBS, 3 × 5 min) and covered with a drop of antifade solution, and the slides were mounted with coverslips. Cell iPLA2beta immunofluorescence was visualized by confocal microscopy at an excitation wavelength of 550 nm and an emission wavelength of 570 nm.

Visualization of nuclear localization of iPLA2beta by dual-labeling fluorescence. To examine whether the iPLA2beta protein was accumulating in the nuclear region, after incubation with Cy3, the cells were washed (PBS, 2 × 5 min) and further incubated (12 min, RT) with bisbenzimide (1:20,000), a fluorescent DNA stain that intercalates in A-T regions of DNA. The cells were washed (PBS, 3 × 5 min) and covered with a drop of antifade solution, and the slides were mounted with coverslips. Rhodamine and DAPI filters attached to a Nikon Eclipse TE 300 Inverted Scope were used to visualize Cy3 (iPLA2beta , red) and bisbenzimide (nuclei, blue) fluorescences, respectively, separately and in combination.

Effects of glucose, IBMX, and FSK stimulation on insulin secretion and iPLA2beta protein localization. At confluence, iPLA2beta -overexpressing cells were detached from T75 flasks, and aliquots of cells (~2 · 104) were seeded in multichambered glass slides and allowed to attach overnight. The culture medium was then removed, and the cells were washed twice in Krebs-Ringer buffer (KRB), pH 7.3, containing (in mM): 115 NaCl, 24 NaHCO3, 5 KCl, 1 MgCl2, 25 HEPES, 1 glucose, and 0.10% BSA and were incubated in the same medium (1 h, 37°C, under an atmosphere of 95% air-5% CO2). The medium was then removed and replaced with KRB medium containing glucose (0-20 mM) without or with IBMX (100 µM) or FSK (2.5 µM). The cells were further incubated (0-60 min, 37°C, under an atmosphere of 95% air-5% CO2). At the end of the incubation period, the medium was removed for measurement of insulin by radioimmunoassay (65), and the cells were harvested for iPLA2beta enzymatic activity assays, as we have described.

In vitro phosphorylation of iPLA2beta . Islet iPLA2beta was overexpressed in and purified from sf9 insect cells by use of sequential (DEAE anion-exchange, ATP-affinity, and calmodulin-affinity) column chromatography, as previously described (49). Aliquots of the purified protein were incubated (30 min, 37°C) with buffer (10 mM Tris, 10 mM MgAc, 4 mM EGTA, pH 7.20), cPKA (10 U), and 10 µM [gamma -32P]ATP (total volume 110 µl), without and with either PKAI (25 µM), the tyrosine phosphorylation inhibitor genistein (100 µM), or staurosporine (100 nM), a nonspecific inhibitor of phosphorylation. After the assay period, the samples were immediately diluted in sample buffer and boiled. Aliquots of the samples were then analyzed by SDS-PAGE (7.5%), and the protein from gels was transferred onto Immobilon-P PVDF membrane. The transferred 32P-labeled proteins were visualized by autoradiography. Subsequently, the same blot was processed for iPLA2beta immunoaffinity analyses, as we have described.

In vitro glycosylation of purified iPLA2beta . To detect the presence of O-GlcNac modification of the iPLA2beta , protein transfer of radiolabeled galactose onto the iPLA2beta protein in the presence of beta -D-galactosyltransferase was examined using reagents supplied in the GalNac kit. Aliquots of purified iPLA2beta or standard hen egg albumin were incubated (4°C, 3 h, total volume of 50 µl) with reaction buffer B, consisting of 5 mM MnCl2, UDP [3H]galactose (sp act 5-20 mCi/mmol), and beta -D-galactosyltransferase (0.025 U/ml). The reaction was terminated by the addition of sample buffer. Aliquots of the reaction mixtures were analyzed by SDS-PAGE, and the gel was fixed (30% MeOH/7% HAc, 15 min), soaked in Enhancer solution (30 min) and then in 10% glycerol (30 min), dried (80°C, 1 h), and cooled (10 min). Then followed visualization of labeled protein by autoradiography.

Effects of inhibition of PKA and O-glycosylation on insulin secretion, iPLA2beta protein localization, and enzymatic activity. To examine whether phosphorylation or glycosylation events contribute to iPLA2beta protein translocation in the cell, cellular permeable inhibitors of PKA (H-89) and of O-glycosylation (BG) were utilized. INS-1 cells overexpressing iPLA2beta were seeded in 24-well plates (for insulin secretion assays), flasks (for enzymatic activity assays), or glass slides (for immunofluorescence assays), and were pretreated with either H-89 (10-20 µM, 60 min) or BG (2-4 mM, 24 h), prepared in DMSO. The inhibitors were also included during stimulation of the cells with glucose (2 mM) in the absence and presence of FSK (2.5 µM). Control cells were incubated with the vehicle DMSO alone. After the incubation period, the medium was collected for measurement of insulin content, and the cells were processed either for enzymatic activity assays or immunofluorescence analyses.

Statistical analyses. The data were converted to mean ± SE values where appropriate, and the Student's t-test was applied to determine significant differences (at P < 0.05) between two groups.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Enzymatic activity and protein expression of iPLA2beta in transfected INS-1 cells. To verify expression of active iPLA2beta protein in INS-1 cells transfected with iPLA2beta cDNA, homogenates were prepared from INS-1 cells transfected with either an empty retroviral (V) construct or with an iPLA2beta cDNA (I)-containing construct. Aliquots of protein from these fractions were used to determine iPLA2beta activity, by radiochemical enzymatic assay, and iPLA2beta protein expression, by immunoaffinity analyses. As expected, homogenates prepared from INS-1 cells transfected with iPLA2beta cDNA expressed higher activity (nearly 10-fold) than cells transfected with the V construct (Fig. 1A). The increased expression of activity was reflected by the higher expression of iPLA2beta protein in the INS-1 cells transfected with iPLA2beta cDNA relative to V cells (Fig. 1B). These results confirm that transfection of INS-1 cells with iPLA2beta cDNA results in higher expression of catalytically active iPLA2beta .


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 1.   Enzymatic activity and protein expression of cytosolic calcium-independent phospholipase A2 (iPLA2beta ) in insulin-secreting (INS-1) cells. A: enzymatic activity analyses. Subcellular fractions were prepared from INS-1 cells transfected with either an empty vector (V) or a vector containing iPLA2beta cDNA (I). Aliquots of cellular homogenate protein were incubated (37°C, 5 min) in the presence of EGTA (5 mM) and [14C]PLPC, and the hydrolyzed [14C]linoleate was quantified by liquid scintillation spectrometry. The calculated specific iPLA2beta enzymatic activity is presented. Data are means ± SE of specific iPLA2beta enzymatic activity (pmol · mg protein-1 · min-1) in each group (n = 6). B: immunoaffinity analyses. Aliquots of homogenates prepared from cells infected with vector only (V) or with iPLA2beta cDNA (I) were analyzed by SDS-PAGE, and the proteins were transferred onto Immobilon-P polyvinylidene difluoride (PVDF) membrane. The electroblot was subsequently probed with antibodies directed against iPLA2beta protein.

Distribution of iPLA2beta in overexpressing INS-1 cells. Immunoaffinity, activity, and immunofluorescence analyses were next used to examine in greater detail the distribution of iPLA2beta within the INS-1 cells (Fig. 2). Cellular fractions prepared from iPLA2beta -overexpressing INS-1 cells, as described in METHODS, were used in these analyses. As illustrated in Fig. 2A, left, abundant immunoreactive iPLA2beta protein was evident in the CH, NN (containing cytosol), and NE fractions, with low iPLA2beta protein content evident in the P fraction. The specificity of the antibody affinity for iPLA2beta was then examined after incubations with secondary antibody alone (Fig. 2A, lane 1), preimmune IgG (lane 2), or piPLA2beta antibodies plus antigenic peptide sequences of iPLA2beta against which the antibodies were generated (lane 3). Under all three conditions, no signal for the iPLA2beta protein was detected. These findings confirm that the piPLA2beta antibodies generated by Research Genetics strategy are specific for the iPLA2beta protein. Figure 2A, right, reflects iPLA2beta enzymatic activity in the corresponding INS-1 cell fractions and illustrates an abundance of iPLA2beta catalytic activity in the nuclear fraction.


View larger version (36K):
[in this window]
[in a new window]
 
Fig. 2.   Immunoaffinity, activity, and immunofluorescence evidence for expression and localization of iPLA2beta in transfected INS-1 cells. A: immunoaffinity and activity analyses. Cellular fractions, prepared from iPLA2beta -overexpressing INS-1 cells, were analyzed by SDS-PAGE, and the proteins were transferred onto Immobilon-P PVDF membrane. The electroblot was probed with either the secondary peroxidase-conjugated goat anti-rabbit IgG antibody alone (lane 1), preimmune IgG alone (lane 2), iPLA2beta antibodies (CH, cellular homogenate; NN, nonnuclear homogenate; NE, nuclear extract; and P, particulate fraction), or piPLA2beta antibodies plus iPLA2beta peptide antigens against which the piPLA2beta antibodies were generated (lane 3). B: immunofluorescence analyses. INS-1 cells overexpressing iPLA2beta seeded in chambered glass slides were fixed, permeabilized, and probed with either iPLA2beta antibodies (a), preimmune IgG alone (b), or piPLA2beta antibodies plus iPLA2beta peptide antigens (c). The fluorescent Cy3-conjugated AffiniPure goat anti-rabbit IgG was used as the secondary antibody to visualize iPLA2beta in the cells by confocal microscopy.

The piPLA2beta antibodies were next used to examine localization of the iPLA2beta protein within the INS-1 cells by immunofluorescence analyses. As illustrated in Fig. 2B, in the presence of piPLA2beta antibodies (a) a diffuse iPLA2beta fluorescence signal is detected in the cytosol of cells along with a ring of fluorescence in the nuclear region. No fluorescence signal, however, is evident when preimmune IgG is used as the primary probe (b). The iPLA2beta protein fluorescence is also completely neutralized when the cells are treated with piPLA2beta antibodies in the presence of the peptide antigens (c). These findings confirm that the piPLA2beta antibodies have specific affinity for the iPLA2beta protein in both isolated cellular fractions and in intact cells.

Immunofluorescence analyses of iPLA2beta distribution in INS-1 cells after stimulation with glucose and IBMX. To examine whether the iPLA2beta protein undergoes translocation under conditions that promote insulin secretion, parental INS-1 cells and INS-1 cells transfected with iPLA2beta cDNA were stimulated with glucose and IBMX, either alone or in combination. The cells were then sequentially fixed, permeabilized, and incubated with piPLA2beta antibodies followed by Cy3 fluorescent secondary antibody. Localization of iPLA2beta fluorescence was then visualized by confocal microscopy. Fluorescence recordings for iPLA2beta in parental cells after a 60-min stimulation period with glucose in the absence and presence of IBMX are presented in Fig. 3. In the presence of no added glucose or IBMX, iPLA2beta fluorescence is diffuse (A). At 2 mM glucose, the iPLA2beta fluorescence appears as a ring (B) in the nuclear region. Higher glucose concentrations result in more prominent fluorescence, which is distributed throughout the cytoplasm and nuclear regions (C and D). Addition of IBMX by itself results in distinct iPLA2beta fluorescent ring formation (E), which becomes more diffuse with increasing concentrations of glucose (F-H).


View larger version (73K):
[in this window]
[in a new window]
 
Fig. 3.   Immunofluorescence analyses of iPLA2beta distribution in parental INS-1 cells after stimulation with glucose ± IBMX. Parental INS-1 cells were seeded on chambered glass slides and incubated in KRB medium (1 ml) containing glucose (G) without and with IBMX (100 µM) for 1 h at 37°C under 95% air-5% CO2 atmosphere. At the end of the incubation period, medium was removed, and the cells were washed with PBS, fixed, permeabilized, and incubated (O/N) with piPLA2beta antibodies. Cells were then incubated with fluorescent Cy3-conjugated AffiniPure goat anti-rabbit IgG to visualize the iPLA2beta protein by confocal microscopy. A-D: 0, 2, 10, or 20 mM G, respectively. E-H: IBMX in the presence of 0, 2, 10, or 20 mM glucose, respectively.

In contrast to parental cells, under basal conditions, a diffuse iPLA2beta fluorescence distribution in the cytosol is accompanied by distinct rings of iPLA2beta fluorescence around the nuclear region of INS-1 cells transfected with iPLA2beta cDNA (Fig. 4A). Increases in the glucose concentration to 2 mM result in more distinct ring formation (B), reflecting increased accumulation of iPLA2beta in the nuclear region. Higher glucose concentrations, however, do not further promote increases in nuclear fluorescence of iPLA2beta (C and D). Addition of IBMX alone (E) results in robust increases in iPLA2beta fluorescence around the nuclear region. The intense iPLA2beta fluorescence with IBMX is modestly increased in the presence of glucose (F-H).


View larger version (95K):
[in this window]
[in a new window]
 
Fig. 4.   Immunofluorescence analyses of iPLA2beta distribution in overexpressing INS-1 cells after stimulation with glucose and IBMX. A-H: INS-1 cells overexpressing iPLA2beta were treated as described in Fig. 3. A-D: 0, 2, 10, or 20 mM G, respectively. E-H: IBMX (100 µM) in the presence of 0, 2, 10, or 20 mM G, respectively. I-K: iPLA2beta fluorescence was monitored at 15, 30, and 60 min, respectively, after treatment with IBMX. L: time-dependent insulin secretion from IBMX-stimulated iPLA2beta -overexpressing INS-1 cells. Parental (open circle ) and iPLA2beta transfected () INS-1 cells were incubated as described in Fig. 3, with 2 mM G, alone or with IBMX (I, 100 µM). Insulin content in the medium was determined by RIA. Stimulated increases in insulin secretion are plotted relative to secretion in the absence of glucose at time 0. Data are means of 2-6 individual measurements.

To monitor temporal changes in the distribution of the iPLA2beta protein during stimulation, INS-1 cells transfected with iPLA2beta cDNA were examined at various times during the 60-min exposures to either glucose or IBMX. Glucose alone (data not shown) at all concentrations promotes nuclear accumulation of iPLA2beta after 30 min of stimulation, which is reflected by the appearance of fluorescent rings in the nuclear region. However, by 60 min, iPLA2beta fluorescence at the nuclear perimeter disperses, and the signal becomes diffuse within the nuclei and in the cytosol. The relatively little effect of increasing concentrations of glucose on iPLA2beta distribution is associated with a diminished capacity to secrete insulin, relative to iPLA2beta -overexpressing cells, over the same range of glucose concentrations. This is illustrated in Fig. 4 (open circle  data points in L), where secretion after a 60-min incubation period with 2 mM glucose in the absence and presence of IBMX is presented.

Stimulation of the cells with IBMX, in contrast, promotes increases in time-dependent accumulation of iPLA2beta fluorescence in the nuclear region (Fig. 4, I-K), which is reflected by the appearance of a diffused pattern by 15 min (I), relative to distribution at 0 min (A). After a 30-min period of stimulation with IBMX, reformation of distinct iPLA2beta fluorescent rings around the nuclear region is evident (J), and subsequently becomes more intense by 60 min (K). The IBMX-induced distribution of iPLA2beta is found to be similar in the absence and presence of glucose (2 mM). The time-dependent IBMX-induced accumulation of iPLA2beta in the nuclear region of iPLA2beta overexpressing INS-1 cells correlates well with the amplified temporal increase in IBMX-induced insulin secretion ( data points in L) relative to parental responses (open circle , L).

Visualization of nuclear accumulation of iPLA2beta by dual-signal immunofluorescence. To verify that the stimulated iPLA2beta signal was indeed accumulating in the nuclear region, a dual-labeling fluorescence protocol was used. INS-1 cells overexpressing iPLA2beta were stimulated with either glucose (2 mM) or IBMX (100 µM) alone and subsequently processed for dual-fluorescence analyses. The iPLA2beta protein is visualized with Cy3 red fluorescence and the nuclear region with bisbenzimide blue fluorescence. An overlap of the two fluorescences would yield a pinkish hue and reflect the appearance of the iPLA2beta protein in the nuclear region. This is illustrated in Fig. 5. In A, only the iPLA2beta fluorescence with 2 mM glucose is visualized; in B, only the nuclear region in the corresponding cells is visualized. When both fluorescences are simultaneously monitored (C), a clear separation of the red iPLA2beta from the blue nuclear fluorescence is evident. Figure 5D depicts the higher-intensity iPLA2beta fluorescence in the presence of IBMX alone, and Fig. 5E reveals the nuclear region in the corresponding cells. When both fluorescences are simultaneously monitored in the IBMX-stimulated cells (F), the nuclear blue region is replaced by a pinkish coloring from the overlap of iPLA2beta and the nuclear region, suggesting that, in the presence of IBMX, the iPLA2beta protein accumulates in the nuclear region.


View larger version (66K):
[in this window]
[in a new window]
 
Fig. 5.   Visualization of nuclear accumulation of iPLA2beta by dual-signal immunofluorescence. INS-1 cells overexpressing iPLA2beta were incubated with either glucose (G, 2 mM) or IBMX (100 µM) for 1 h, as described in Fig. 4, and processed for immunofluorescence analyses, as described in Fig. 3. After incubation with the Cy3 secondary antibody, cells were incubated with bisbenzimide [12 min at room temperature (RT)]. iPLA2beta fluorescence (red) was visualized with a rhodamine filter, and the nuclear fluorescence (blue) was visualized with a DAPI filter with a Nikon Inverted Scope. A-C, 2 G; D-F, 100 µM IBMX. A and D: Cy3 (iPLA2beta ) fluorescence; B and E: bisbenzimide (nuclear region) fluorescence; C and F: dual Cy3 and bisbenzimide fluorescences.

Verification of purity of nuclear preparation. To determine whether the accumulation of iPLA2beta protein in the nuclear region was accompanied by increases in nuclear iPLA2beta activity, nuclear fractions were prepared from iPLA2beta -overexpressing INS-1 cells according to the procedure of Schreiber et al. (41). Because we have previously observed the presence of iPLA2beta activity in mitochondrial and plasma membrane compartments of beta -cells (14), the possibility that the nuclear preparation is contaminated with these cellular components was first assessed. As illustrated in Fig. 6, mitochondria-associated cytochrome c oxidase (A) and plasma membrane-associated 5'-nucleotidase (B) activities are enriched in the NN fraction relative to the nuclear fraction. Furthermore, immunoreactive nuclear transcription factor Oct-1 is evident in the nuclear fraction (C) but not in the NN fraction. These findings verify that the NE fraction is not contaminated with mitochondria or plasma membrane and contains nuclei. To verify that the Ca2+-independent PLA2 activity expressed in NN and NE fractions is manifested by the iPLA2beta protein, the ability of ATP to stimulate and of BEL, a suicide inhibitor of iPLA2beta (35), to inhibit activity was determined. The beta -cell iPLA2beta enzymatic activity has previously been shown to be uniquely sensitive to ATP and BEL (35-37). As demonstrated in Fig. 6D, the enzymatic activity expressed in both fractions is found to be stimulated by ATP and completely inhibited by BEL; arrows indicate absence of measurable activity. These findings therefore confirm the presence of iPLA2beta protein in the INS-1 cell fractions of interest.


View larger version (38K):
[in this window]
[in a new window]
 
Fig. 6.   Verification of purity of nuclear preparation and presence of of iPLA2beta activity in the nuclear fraction. Cellular fractions from the iPLA2beta -overexpressing INS-1 cells were prepared, as described in METHODS, and were assayed for mitochondria-associated cytochrome c oxidase (A) and plasma membrane-associated 5'-nucleotidase (B) activities and nuclear Oct-1 immunoaffinity (C). D: iPLA2beta enzymatic activity in the absence (Con) and presence of either ATP (10 mM) or BEL (10 µM) in the nonnuclear and nuclear fractions, respectively. Data in A, B, and D represent means ± SE of specific enzymatic activities of cytochrome c oxidase (mU/mg protein), 5'-nucleotidase (pmol · mg protein-1 · min-1), and iPLA2beta (pmol · mg protein-1 · min-1), respectively (n = 4). *P < 0.05, ATP-treated groups significantly different from control groups. Arrows, complete inhibition of iPLA2beta enzymatic activity after BEL treatment. CH, cellular homogenate; NN, nonnuclear homogenate; NH, nuclear homogenate; NE, nuclear extract; P, particulate fraction.

Stimulated accumulation of nuclear iPLA2beta enzymatic activity. To examine more directly the influence of cAMP-elevating agents on the distribution of iPLA2beta in overexpressing cells, the adenylate cyclase stimulator FSK was utilized in subsequent studies. Hence, iPLA2beta activity was determined in the NN and NE fractions after treatment of iPLA2beta -overexpressing INS-1 cells with glucose (2 mM) in the absence and presence of FSK. As illustrated in Fig. 7A, NN-associated specific iPLA2beta activity is relatively unchanged under 2 G ± FSK conditions. This is confirmed in immunoaffinity analyses, which reveals no significant change in the iPLA2beta content in this fraction (data not presented). In contrast, nuclear-associated iPLA2beta activity increases only modestly (Fig. 7B) with 2 G, but significantly (nearly 2-fold) with the addition of FSK. Such an increase in activity is reflected by increased accumulation of the iPLA2beta protein (Fig. 7C) in the NE fraction after stimulation with FSK.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 7.   Localization of INS-1 cell iPLA2beta activity after stimulation with forskolin (FSK). Nonnuclear and nuclear fractions prepared from the iPLA2beta -overexpressing INS-1 cells were assayed for iPLA2beta enzymatic activity, as described in METHODS. Enzymatic iPLA2beta activity was determined in the nonnuclear (A) and nuclear (B) fractions after stimulation of cells with glucose (2 mM) in the absence and presence of FSK (2.5 µM). Data are means ± SE of specific iPLA2beta enzymatic activity (pmol · mg protein-1 · min-1) determined in each fraction (n = 12-16). *P < 0.05, significantly different from 0 G group. C: immunoaffinity analyses of iPLA2beta in the nuclear fraction after stimulation of cells with 2 G ± FSK.

Potential phosphorylation and glycosylation of iPLA2beta . Previous reports suggest that phosphorylation stimulates nuclear association of catalytic (c) PLA2 (13, 40), and that cytosolic and nuclear proteins undergo stimulated glycosylation (7, 15). In view of this, it was of interest to examine the potential involvement of these two processes in nuclear accumulation of iPLA2beta in INS-1 cells. Elevations in cAMP, as with FSK, induce cAMP-dependent PKA-induced phosphorylation of serine and threonine residues of proteins (8). As such, the ability of cPKA to phosphorylate iPLA2beta was examined. Treatment of purified iPLA2beta with cPKA promotes incorporation of 32P into the protein (Fig. 8A, top, lane 1). Such PKA-induced phosphorylation of iPLA2beta is completely blocked by a specific peptide inhibitor of PKA (PKAI, lane 2) and by the nonspecific inhibitor of phosphorylation, staurosporine (lane 5) but not by genestein (lane 3), which inhibits tyrosine phosphorylation. As shown (bottom), immunoaffinity analysis of the same blot verifies that all lanes are loaded with similar iPLA2beta protein amounts. As illustrated in Fig. 8B, incubation of purified iPLA2beta with galactosyltransferase results in glycosylation of the protein. These findings raise the possibility that beta -cell iPLA2beta is a candidate for phosphorylation and glycosylation.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 8.   In vitro phosphorylation and O-glycosylation of purified iPLA2beta . A: phosphorylation. Aliquots of islet iPLA2beta purified from sf9 cells were incubated (30 min, 37°C) with catalytic PKA (cPKA, 10 U) and [gamma -32P]ATP (10 µM), without and with inhibitors PKAI (25 µM), genistein (100 µM), or staurosporine (100 nM). Samples were then analyzed by SDS-PAGE, and proteins were transferred onto Immobilon-P PVDF membrane. 32P-labeled proteins were then visualized by autoradiography, and the same blots were subsequently used for immunoaffinity assays. Lanes 1 and 4, cPKA alone; lane 2, cPKA + PKAI; lane 3, cPKA + genistein; lane 5, cPKA + staurosporine. B: O-glycosylation. Aliquots of islet iPLA2beta purified from sf9 cells and hen egg albumin standard were incubated (3 h, 4°C) with UDP [3H]galactose and beta -D-galactosyltransferase (0.025 U/ml). The reaction was terminated with addition of sample buffer, and the mixture was analyzed by SDS-PAGE. The gel was then processed for visualization of 3H-labeled protein by autoradiography. Lane 1, 1× protein concn; 2, 2× protein concn.

Involvement of protein phosphorylation and glycosylation during insulin secretion. To examine whether protein phosphorylation or glycosylation has a role during stimulation of insulin secretion from iPLA2beta -overexpressing INS-1 cells, the effects of H-89 and BG, cell-permeable specific inhibitors of PKA (50) and O-glycosylation (20), respectively, were examined. As shown in Fig. 9, insulin secretion from the overexpressing INS-1 cells is modestly elevated in the presence of 5 mM glucose alone, but robustly amplified with additions of cAMP-elevating agents IBMX and FSK. Both H-89 and BG significantly attenuate the IBMX- and FSK-induced responses, suggesting that phosphorylation and glycosylation events participate in insulin- secretory responses amplified by cAMP-elevating agents.


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 9.   Insulin secretion from iPLA2beta -overexpressing INS-1 cells. Cells were seeded on chambered glass slides and incubated in KRB medium containing glucose, without and with IBMX (100 µM) or FSK (2.5 µM) in the absence (Con) and presence of either H-89 (10-20 µM) or BG (2-4 mM) for 1 h at 37°C under 95% air-5% CO2 atmosphere. At the end of the incubation period, insulin content in the medium was determined by RIA. Stimulated increases in insulin secretion are plotted as means ± SE relative to secretion in the stimulant (n = 10-16). dagger Significantly different from 0 G group; #significantly different from corresponding groups treated with glucose alone; *inhibitor-treated groups significantly different from control groups, all P < 0.05.

Effects of inhibitors of phosphorylation and glycosylation on iPLA2beta localization. Immunofluorescence analyses were performed next to examine whether inhibition of protein phosphorylation or glycosylation affects stimulated nuclear accumulation of iPLA2beta . As illustrated in Fig. 10, relative to the 0 G condition (A), presence of 2 G alone promotes appearance of a modest ring-like accumulation of iPLA2beta in the nuclear region (B). Neither H-89 (C) nor BG (D) appears to affect such accumulation. FSK alone (E), similar to IBMX (Fig. 4), increases nuclear accumulation of the iPLA2beta protein, and addition of 2 G further promotes this association into ring-like formation (F). After treatment with H-89 (G) or BG (H), FSK-amplified increases in nuclear immunofluorescence of iPLA2beta are markedly reduced. When iPLA2beta enzymatic activity was determined in the corresponding groups, FSK-stimulated increase in nuclear iPLA2beta activity was found to return to basal levels (Fig. 11). Collectively, these findings suggest that phosphorylation and glycosylation events participate in stimulated nuclear accumulation of iPLA2beta in INS-1 beta -cells.


View larger version (80K):
[in this window]
[in a new window]
 
Fig. 10.   Effects of H-89 and BG on forskolin-stimulated nuclear accumulation of iPLA2beta immunofluorescence. iPLA2beta -overexpressing INS-1 cells were incubated with glucose (2 mM) without and with FSK (2.5 µM) for 1 h in the absence and presence of inhibitors and processed for immunofluorescence analyses, as described in Fig. 3. A: 0 G; B: 2 G; C: 2 G + H-89; D: 2 G + BG; E, 0 G + FSK; F, 2 G + FSK; G: 2 G + FSK + H-89; H: 2 G + FSK + BG.



View larger version (44K):
[in this window]
[in a new window]
 
Fig. 11.   Effects of H-89 and BG inhibitors of forskolin-stimulated nuclear accumulation of iPLA2beta enzymatic activity. Nuclear fractions were prepared from iPLA2beta -overexpressing INS-1 cells after glucose (2 mM), without and with FSK (2.5 µM) treatments in the absence and presence of inhibitors, as described in METHODS. Fractions were then assayed for iPLA2beta activity. Data are means ± SE of %changes in specific iPLA2beta enzymatic activity relative to that measured in the 2 mM glucose-treated groups (n = 8-12). #Significantly different from 2 G group; *significantly different from 2 G+FSK group, P < 0.05.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Potential functions proposed for the beta -isoform of calcium-independent Group VIA PLA2 (iPLA2beta ) include generation of substrate for eicosanoid synthesis (28), membrane degradation during apoptosis (2), regulation of phosphatidylcholine composition and content (4, 5), and involvement in signaling events (19, 46).

Studies in murine P388D1 macrophage-like cells have led to the suggestion that iPLA2beta participates in phospholipid remodeling and therefore serves a housekeeping role (4). In that study, they observed that inhibition of iPLA2beta activity with either BEL or with antisense oligonucleotide treatments led to suppressed incorporation of arachidonic acid into the phosphatidylcholine pool and also reduced the generation of lysophosphatidylcholine (4). Subsequent studies in our laboratory, however, indicate that suppression of iPLA2beta inhibits insulin secretion without affecting arachidonic acid incorporation into the beta -cell phosphatidylcholine pool (35). Furthermore, when the proposed role of iPLA2beta in phospholipid remodeling was examined in INS-1 insulinoma cells overexpressing iPLA2beta , the rate or extent of arachidonic acid incorporation into INS-1 cell phospholipids was not found to be altered, although the insulin-secretory response was markedly greater in the iPLA2beta -overexpressing INS-1 cells (22). These findings strongly suggest that the iPLA2beta enzyme manifests a signaling, rather than a housekeeping, role in beta -cells.

The iPLA2beta -overexpressing INS-1 cells studied in the present study were found to be more sensitive to cAMP-elevating agents than parental INS-1 cells. Increasing concentrations of glucose produced only modest increases in insulin secretion, but IBMX (a phosphodiesterase inhibitor) and FSK (an adenyl cyclase activator) promoted robust increases in the insulin secretory response. Attenuated responses to glucose alone have previously been recognized in native beta -cells that are stimulated after separation from the non-beta -cell population of pancreatic islets (29). Glucose stimulation of isolated islet beta -cells promotes only minor increases in insulin secretion, but costimulation with cAMP-elevating agents or reintroduction of non-beta -cells restores the insulin-secretory capacity (31). The ability of cAMP-elevating agents to amplify glucose-stimulated insulin secretion has also been noted in insulinoma cell lines (32). These observations highlight the requirement for maintaining a critical level of cAMP for glucose-stimulated insulin secretion.

Overexpression of iPLA2beta in INS-1 cells promotes accumulation of the enzyme in the nuclear region, even under basal conditions. Additions of increasing concentrations of glucose do not influence this accumulation significantly, but cAMP-elevating agents dramatically enhance accumulation of iPLA2beta in the nuclear region, as reflected by immunofluorescence, immunoaffinity, and enzymatic activity analyses. This effect is time dependent and very closely correlates with the secretory response seen in the iPLA2beta -overexpressing cells. Such a stimulated translocation of iPLA2beta provides further evidence to support its potential role in signaling during secretory events.

Nuclear association of iPLA2beta induced by cAMP-elevating agents in INS-1 cells is of interest, because glucose promotes beta -cell insulin secretion and proliferation, and glucose-induced INS-1 cell mitogenesis is cAMP dependent (18). As membranes of the nucleus and endoplasmic reticulum (ER) are contiguous (17, 40), perinuclear accumulation of iPLA2beta is consistent with association with a subcellular compartment that is likely to include ER (17). The beta -cell ER is known to contain an abundance of arachidonate-containing plasmenylethanolamine molecular species (33), and products of PLA2 action induce Ca2+ release from beta -cell ER (48), which is thought to participate in induction of insulin secretion (10). cAMP-mediated increases in cytosolic [Ca2+], via Ca2+ entry and mobilization of intracellular Ca2+ stores (11, 17, 51), and sensitization of the exocytotic apparatus to Ca2+ (1) are among the mechanisms by which cAMP augments the insulin- secretory responses. In view of earlier observations suggesting involvement of arachidonic acid, in particular of arachidonic acid hydrolyzed from plasmenylethanolamine molecular species (33, 34) and of its metabolites in the beta -cell secretory process (45), the nuclear association of iPLA2beta after stimulation with cAMP-elevating agents might be consistent with a process that results in the generation of arachidonic acid in close proximity to enzymes, which catalyze eicosanoid generation, localized in the nuclear envelope and ER (27, 39).

The nuclear association of cPLA2 after cellular stimulation with the calcium ionophore A-23187 has been reported to be associated with increased phosphorylation of the cPLA2 (13, 40). Although nuclear accumulation of iPLA2beta has been reported during myocardial ischemia (47), it is not known whether nuclear accumulation is associated with phosphorylation of the iPLA2beta . Because stimulation with IBMX or FSK increases cAMP content in INS-1 cells (22), it might be speculated that, as a consequence, a cAMP-dependent PKA, which phosphorylates serine and threonine residues, is activated and that the iPLA2beta serves as a candidate for PKA-catalyzed phosphorylation. The likelihood that the iPLA2beta becomes associated with the nuclear region is enhanced by the recognition of a bipartite nuclear localization sequence (511KREFGEHTKMTDVKKPK527) in the deduced amino acid sequence of iPLA2beta (24) that is similar to the sequence in other nuclear proteins (10, 38).

To examine the possibility that iPLA2beta can undergo PKA-stimulated phosphorylation, islet iPLA2beta overexpressed in sf9 cells and purified was incubated with cPKA. Under the conditions studied, PKA promotes phosphorylation of iPLA2beta in a protein concentration-dependent manner (data not shown). Such phosphorylation is completely blocked by a specific peptide inhibitor of PKA and by the general inhibitor of phosphorylation staurosporine, but not by genistein, which inhibits tyrosine phosphorylation. These findings raise the possibility that iPLA2beta is a candidate for PKA-induced phosphorylation. In view of this, effects of inhibiting cellular PKA activity with H-89 (a cell-permeable inhibitor) on iPLA2beta distribution were examined. The data obtained in the presence of H-89 reveal that FSK-stimulated accumulation of iPLA2beta immunofluorescence in the nuclear region is dramatically attenuated and that this is accompanied by reductions in FSK-stimulated nuclear accumulation of iPLA2beta enzymatic activity and by inhibition of cAMP-elevating agent-stimulated insulin secretion.

Another potential modification of the iPLA2beta protein that might affect its localization is glycosylation, a modification that has been reported with several cytosolic and nuclear proteins (7, 15). Although it is not clear what effect glycosylation of the iPLA2beta protein might have on its function or localization, it might be noted that the iPLA2beta contains a consensus sequence site for O-GlcNacylation. Interestingly, iPLA2beta also contains ankyrin-like repeat sequences (25) that bind tubulin and integral membrane proteins (30), and O-GlcNacylation is involved in neurofilament assembly (16). A number of agents that interfere with microtubule assembly or disassembly inhibit insulin secretion (21). In view of these observations, it might be speculated that glycosylation of the iPLA2beta promotes its binding to the neurofilament processes through the ankyrin-like repeats and facilitates its trafficking into the nuclear region. Such a possibility was examined using purified iPLA2beta and intact cells. Incubation of purified iPLA2beta with galactosyltransferase resulted in an increase in the incorporation of labeled galactose, suggesting that the protein was a candidate for O-GlcNacylation. After treatment of iPLA2beta -overexpressing cells with BG, a cell-permeable competitive inhibitor of galactosaminyltransferase and O-glycosylation, decreases in FSK-stimulated nuclear accumulation of iPLA2beta fluorescence are observed. As with H-89, FSK-stimulated nuclear accumulation of iPLA2beta activity and insulin secretion are also significantly attenuated by BG. Collectively, the data obtained with H-89 and BG raise the possibility that phosphorylation and glycosylation of the iPLA2beta protein itself, or of associated proteins involved in the secretory pathway, might participate in promoting translocation of iPLA2beta to the nuclear region.

In summary, in the present study we report that the beta -cell iPLA2beta becomes associated with the nuclear region under conditions that promote increases in insulin secretion. Posttranslational modifications such as phosphorylation and glycosylation may potentially participate in promoting its association with the nuclear region and facilitate its signaling role in beta -cells. Detailed analyses are still required to identify the sites in the iPLA2beta protein that might be modified during stimulatory conditions, and such studies are currently underway.


    ACKNOWLEDGEMENTS

We thank Karen Green, Dr. William Cruz, and Dr. Burton Wice for their advice on immunofluorescence assays; Sam Smith and Dr. Kevin Yarasheski for their assistance with the cytochrome c oxidase assay; and Alan Bohrer and Dr. Mary Wohltmann for their expert technical assistance.


    FOOTNOTES

This research was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-34388, a Career Development Award (to Z. Ma) from the Juvenile Diabetes Foundation (no. 2-1999-55), and a Career Development Award (to S. Ramanadham) from the American Diabetes Association.

Address for reprint requests and other correspondence: S. Ramanadham, Washington Univ. School of Medicine, Dept. of Medicine, Box 8127, 660 S. Euclid Ave., St. Louis, MO 63110 (E-mail: sramanad{at}im.wustl.edu).

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.

10.1152/ajpendo.00165.2001

Received 10 April 2001; accepted in final form 16 November 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alzola, E, Perez-Etxebarria A, Kabre E, Fogarty DJ, Metioui M, Chaib N, Macarulla JM, Matute C, Dehaye JP, and Marino A. Activation by P2X7 agonists of two phospholipases A2 (PLA2) in ductal cells of rat submandibular gland. Coupling of the calcium-independent PLA2 with kallikrein secretion. J Biol Chem 273: 30208-30127, 1998[Abstract/Free Full Text].

2.   Atsumi, GI, Murakami M, Kojima K, Hadano A, Tajima M, and Kudo I. Distinct roles of two intracellular phospholipase A2s in fatty acid release in the cell death pathway. Proteolytic fragment of type IVA cytosolic phospholipase A2 alpha inhibits stimulus-induced arachidonate release, whereas that of type VI Ca2+-independent phospholipase A2 augments spontaneous fatty acid release. J Biol Chem 275: 18248-18258, 2000[Abstract/Free Full Text].

3.   Balboa, MA, Balsinde J, Jones S, and Dennis EA. Identity between the Ca2+-independent phospholipase A2 enzymes from P388D1 macrophages and Chinese hamster ovary cells. J Biol Chem 272: 8576-8590, 1997[Abstract/Free Full Text].

4.   Balsinde, J, Bianco ID, Ackerman EJ, Conde-Frieboes K, and Dennis EA. Inhibition of calcium-independent phospholipase A2 prevents arachidonic acid incorporation and phospholipid remodeling in P388D1 macrophages. Proc Natl Acad Sci USA 92: 8527-8531, 1995[Abstract].

5.   Barbour, SE, Kapur A, and Deal CL. Regulation of phosphatidylcholine homeostasis by calcium-independent phospholipase A2. Biochim Biophys Acta 1439: 77-78, 1999[ISI][Medline].

6.   Coffin, JM, and Varmus HE. Retrovirus. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1996.

7.   Comer, FI, and Hart GW. O-Glycosylation of nuclear and cytosolic proteins. Dynamic interplay between O-GlcNAc and O-phosphate. J Biol Chem 275: 29179-29182, 2000[Free Full Text].

8.   Cross, TG, Scheel-Toellner D, Henriquez NV, Deacon E, Salmon M, and Lord JM. Serine/threonine protein kinases and apoptosis. Exp Cell Res 256: 34-41, 2000[ISI][Medline].

9.   Dennis, EA. The growing phospholipase A2 superfamily of signal transduction enzymes. Trends Biochem Sci 22: 1-2, 1997[ISI][Medline].

10.   Dingwall, C, and Laskey RA. Nuclear targeting sequences---a consensus? Trends Biochem Sci 16: 478-481, 1991[ISI][Medline].

11.   Frodin, M, Sekine N, Roche E, Fillous C, Prentki M, Wollheim CB, and Obberghen EV. Glucose, other secretagogues, and nerve growth factor stimulate mitogen-activated protein kinase in the insulin-secreting beta-cell line, INS-1. J Biol Chem 270: 7882-7889, 1995[Abstract/Free Full Text].

12.   Gijon, MA, and Leslie CC. Phospholipases A2. Cell Develop Biol 8: 297-303, 1997.

13.   Gijon, MA, Spencer DM, Kaiser AL, and Leslie CC. Role of phosphorylation sites and the C2 domain in regulation of cytosolic phospholipase A2. J Cell Biol 145: 1219-1232, 1999[Abstract/Free Full Text].

14.   Gross, RW, Ramanadham S, Kruszka K, Han X, and Turk J. Rat and human pancreatic islet cells contain a Ca2+-independent phospholipase A2 activity selective for hydrolysis of arachidonate which is stimulated by ATP and specifically localized to islet beta -cells. Biochemistry 32: 327-336, 1993[ISI][Medline].

15.   Haltiwanger, RS, Busby S, Grove K, Li S, Mason D, Medina L, Moloney D, Philipsberg G, and Scarrozzi R. O-glycosylation of nuclear and cytoplasmic proteins: regulation analogous to phosphorylation? Biochem Biophys Res Commun 231: 237-242, 1997[ISI][Medline].

16.   Hart, GW. Dynamic O-linked glycosylation of nuclear and cytoskeletal proteins. Annu Rev Biochem 66: 314-335, 1997.

17.   Holz, GG, Leech CA, Heller RS, Castonguay M, and Habener JF. cAMP-dependent mobilization of intracellular Ca2+ stores by activation of ryanodine receptors in pancreatic beta-cells. A Ca2+ signaling system stimulated by the insulinotropic hormone glucagon-like peptide-1-(7-37). J Biol Chem 274: 14147-14156, 1999[Abstract/Free Full Text].

18.   Hugl, SR, White MF, and Rhodes CJ. Insulin-like growth factor I (IGF-I)-stimulated pancreatic beta-cell growth is glucose-dependent. Synergistic activation of insulin receptor substrate-mediated signal transduction pathways by glucose and IGF-I in INS-1 cells. J Biol Chem 273: 17771-17779, 1998[Abstract/Free Full Text].

19.   Isenovic, E, and LaPointe MC. Role of Ca(2+)-independent phospholipase A(2) in the regulation of inducible nitric oxide synthase in cardiac myocytes. Hypertension 35: 249-254, 2000[Abstract/Free Full Text].

20.   Kuan, SF, Byrd JC, Basbaum C, and Kim YS. Inhibition of mucin glycosylation by aryl-N-acetyl-alpha -galactosaminides in human colon cancer cells. J Biol Chem 264: 19271-19277, 1989[Abstract/Free Full Text].

21.   Lacy, PE, Klein NJ, and Fink CJT Effect of cytochalasin B on the biphasic release of insulin in perifused rat islets. Endocrinology 92: 1458-1468, 1973[ISI][Medline].

22.   Ma, Z, Ramanadham S, Bohrer A, Wohltmann M, Zhang S, and Turk J. Studies of insulin secretory responses and of arachidonic acid incorporation into phospholipids of stably transfected insulinoma cells that overexpress Group VIA phospholipase A2 (iPLA2beta ) indicate a signalling rather than a housekeeping role for iPLA2beta . J Biol Chem 276: 13198-13208, 2001[Abstract/Free Full Text].

23.   Ma, Z, Ramanadham S, Kempe K, Chi XS, Ladenson JL, and Turk J. Pancreatic islets express a Ca2+-independent phospholipase A2 enzyme that contains a repeated structural motif homologous to the integral membrane protein binding domain of ankyrin. J Biol Chem 272: 11118-11127, 1997[Abstract/Free Full Text].

24.   Ma, Z, and Turk J. Molecular biology of group VIA Ca2+-independent phospholipase A2. Prog Nucl Acid Mol Biol 67: 1-33, 2001[ISI].

25.   Ma, Z, Wang X, Nowatzke W, Ramanadham S, and Turk J. Human pancreatic islets express mRNA species encoding two distinct catalytically active isoforms of group VI phospholipase A2 (iPLA2) that arise from an exon-skipping mechanism of alternative splicing of the transcript from the iPLA2 gene on chromosome 22q13.1. J Biol Chem 274: 9607-9616, 1999[Abstract/Free Full Text].

26.   Mancuso, DJ, Jenkins CM, and Gross RW. The genomic organization, complete mRNA sequence, cloning, and expression of a novel human intracellular membrane-associated calcium-independent phospholipase A(2). J Biol Chem 275: 9937-9945, 2000[Abstract/Free Full Text].

27.   Morita, I, Schindler M, Regier MK, Otto JC, Hori T, DeWitt DL, and Smith WL. Different intracellular locations for prostaglandin endoperoxide H synthase-1 and -2. J Biol Chem 270: 10902-10908, 1995[Abstract/Free Full Text].

28.   Murakami, M, Kambe T, Shimbara S, and Kudo I. Functional coupling between various phospholipase A2s and cyclooxygenases in immediate and delayed prostanoid biosynthetic pathways. J Biol Chem 274: 3103-3115, 1999[Abstract/Free Full Text].

29.   Owada, S, Larsson O, Arkhammar P, Katz AI, Chibalin AV, Berggren PO, and Bertorello AM. Glucose decreases Na+,K+-ATPase activity in pancreatic beta-cells. An effect mediated via Ca2+-independent phospholipase A2 and protein kinase C-dependent phosphorylation of the alpha-subunit. J Biol Chem 274: 2000-2008, 1999[Abstract/Free Full Text].

30.   Peters, LL, and Lux SE. Ankyrins: structure and function in normal cells and hereditary spherocytes. Semin Hematol 30: 85-118, 1993[ISI][Medline].

31.   Pipeleers, DG, Schuit FC, in't Veld PA, Maes E, Hooghe-Peters EL, Van De Winkel M, and Gepts W. Interplay of nutrients and hormones in the regulation of insulin release. Endocrinology 117: 824-833, 1985[Abstract].

32.   Poitout, V, Olson LK, and Robertson RP. Insulin-secreting cell lines: classification, characteristics and potential applications. Diabetes Metab (Paris) 22: 7-14, 1996[ISI][Medline].

33.   Ramanadham, S, Bohrer A, Gross RW, and Turk J. Mass spectrometric characterization of arachidonate-containing phospholipids in human pancreatic islets and in rat islet beta -cells and subcellular membranes. Biochemistry 32: 13499-13509, 1993[ISI][Medline].

34.   Ramanadham, S, Bohrer A, Mueller M, Jett P, Gross RW, and Turk J. Mass spectrometric identification and quantitation of arachidonate-containing phospholipids in pancreatic islets: prominence of plasmenylethanolamine molecular species. Biochemistry 32: 5339-5351, 1993[ISI][Medline].

35.   Ramanadham, S, Hsu FF, Bohrer A, Ma Z, and Turk J. Studies of the role of Group VI phospholipase A2 in fatty acid incorporation, phospholipid remodeling, lysophosphatidylcholine generation, and secretagogue-induced arachidonic acid release in pancreatic islets and insulinoma cells. J Biol Chem 274: 13915-13927, 1999[Abstract/Free Full Text].

36.   Ramanadham, S, Hsu FF, Zhang S, Bohrer A, and Turk J. Electrospray ionization mass spectrometric analyses of phospholipids from INS-1 insulinoma cells: comparison to pancreatic islets and effects of fatty acid supplementation on phospholipid composition and insulin secretion. Biochim Biophys Acta 1484: 251-266, 2000[ISI][Medline].

37.   Ramanadham, S, Wolf M, Li B, Bohrer A, and Turk J. Glucose-responsitivity and expression of an ATP-stimulatable, Ca2+-independent phospholipase A2 enzyme in clonal insulinoma cell lines. Biochim Biophys Acta 1344: 153-164, 1997[ISI][Medline].

38.   Robbins, J, Dilworth SM, Laskey RA, and Dingwall C. Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence. Cell 64: 615-623, 1991[ISI][Medline].

39.   Rollins, TE, and Smith WL. Subcellular localization of prostaglandin-forming cyclooxygenase in Swiss mouse 3T3 fibroblasts by electron microscopic immuno-cytochemistry. J Biol Chem 255: 4872-4875, 1980[Abstract/Free Full Text].

40.   Schievella, AR, Regiers MK, Smith WL, and Lin LL. Calcium-mediated translocation of cytosolic phospholipase A2 to the nuclear envelope and endoplasmic reticulum. J Biol Chem 270: 30749-30754, 1995[Abstract/Free Full Text].

41.   Schreiber, E, Matthias P, Muller MM, and Schaffner W. Rapid detection of octamer binding proteins with `mini-extracts,' prepared from a small number of cells. Nucleic Acids Res 17: 6419, 1989[ISI][Medline].

42.   Smith, L. Spectrophotometric assay of cytochrome C oxidase. In: Methods of Biochemical Analysis, edited by Glick D.. New York: Wiley-Interscience, 1955, vol. II, p. 427.

43.   Tanaka, H, Takeya R, and Sumimoto H. A novel intracellular membrane-bound calcium-independent phospholipase A(2). Biochem Biophys Res Commun 272: 320-326, 2000[ISI][Medline].

44.   Tang, J, Kriz RW, Wolfman N, Shaffer M, Seehra S, and Jones S. A novel cytosolic calcium-independent phospholipase A2 contains eight ankyrin motifs. J Biol Chem 272: 8567-8575, 1997[Abstract/Free Full Text].

45.   Turk, J, Gross RW, and Ramanadham S. Amplification of insulin secretion by lipid messengers. Diabetes 42: 367-374, 1993[Abstract].

46.   Walev, I, Klein J, Husmann M, Valeva A, Strauch S, Wirtz H, Weichel O, and Bhakdi S. Potassium regulates IL-1 beta processing via calcium-independent phospholipase A2. J Immunol 164: 5120-5124, 2000[Abstract/Free Full Text].

47.   Williams, SD, Hsu FF, and Ford DA. Electrospray ionization mass spectrometry analyses of nuclear membrane phospholipid loss after reperfusion of ischemic myocardium. J Lipid Res 41: 1585-1595, 2000[Abstract/Free Full Text].

48.   Wolf, BA, Turk J, Sherman WR, and McDaniel ML. Intracellular Ca2+ mobilization by AA. Comparison with myo-inositol 1,4,5-trisphosphate in isolated pancreatic islets. J Biol Chem 261: 3501-3511, 1986[Abstract/Free Full Text].

49.   Wolf, MJ, and Gross RW. Expression, purification, and kinetic characterization of a recombinant 80-kDa intracellular calcium-independent phospholipase A2. J Biol Chem 271: 30879-30885, 1996[Abstract/Free Full Text].

50.   Won, JS, Lee JK, and Suh HW. Forskolin inhibits expression of inducible nitric oxide synthase mRNA via inhibiting the mitogen activated protein kinase in C6 cells. Mol Brain Res 89: 1-10, 2001[ISI][Medline].

51.   Yaekura, K, Kakei M, and Yada T. cAMP-signaling pathway acts in selective synergism with glucose or tolbutamide to increase cytosolic Ca2+ in rat pancreatic beta-cells. Diabetes 45: 295-301, 1996[Abstract].


Am J Physiol Endocrinol Metab 282(4):E820-E833
0193-1849/02 $5.00 Copyright © 2002 the American Physiological Society