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*
Sasanka
Ramanadham,
Fong-Fu
Hsu,
Alan
Bohrer,
Zhongmin
Ma, and
John
Turk
From Mass Spectrometry Resource, Division of Endocrinology,
Diabetes, and Metabolism, Department of Medicine, Washington University
School of Medicine, St. Louis, Missouri 63110
 |
ABSTRACT |
An 84-kDa group VI phospholipase
A2 (iPLA2) that does not require
Ca2+ for catalysis has been cloned from Chinese hamster
ovary cells, murine P388D1 cells, and pancreatic islet
-cells. A
housekeeping role for iPLA2 in generating
lysophosphatidylcholine (LPC) acceptors for arachidonic acid
incorporation into phosphatidylcholine (PC) has been proposed because
iPLA2 inhibition reduces LPC levels and suppresses
arachidonate incorporation and phospholipid remodeling in P388D1 cells.
Because islet
-cell phospholipids are enriched in arachidonate, we
have examined the role of iPLA2 in arachidonate incorporation into islets and INS-1 insulinoma cells. Inhibition of
iPLA2 with a bromoenol lactone (BEL) suicide substrate
did not suppress and generally enhanced [3H]arachidonate
incorporation into these cells in the presence or absence of
extracellular calcium at varied time points and BEL concentrations.
Arachidonate incorporation into islet phospholipids involved
deacylation-reacylation and not de novo synthesis, as indicated by experiments with varied extracellular glucose
concentrations and by examining [14C]glucose
incorporation into phospholipids. BEL also inhibited islet cytosolic
phosphatidate phosphohydrolase (PAPH), but the PAPH inhibitor
propranolol did not affect arachidonate incorporation into islet or
INS-1 cell phospholipids. Inhibition of islet iPLA2 did not
alter the phospholipid head-group classes into which
[3H]arachidonate was initially incorporated or its
subsequent transfer from PC to other lipids. Electrospray ionization
mass spectrometric measurements indicated that inhibition of INS-1 cell
iPLA2 accelerated arachidonate incorporation into PC and
that inhibition of islet iPLA2 reduced LPC levels by 25%,
suggesting that LPC mass does not limit arachidonate incorporation into
islet PC. Gas chromatography/mass spectrometry measurements indicated
that BEL but not propranolol suppressed insulin secretagogue-induced
hydrolysis of arachidonate from islet phospholipids. In islets and
INS-1 cells, iPLA2 is thus not required for arachidonate
incorporation or phospholipid remodeling and may play other roles in
these cells.
 |
INTRODUCTION |
Phospholipases A2
(PLA2)1 catalyze
hydrolysis of the sn-2 fatty acid substituent from
glycerophospholipid substrates to yield a free fatty acid and a
2-lysophospholipid (1-7). PLA2 are a diverse group of
enzymes, and the first members to be well characterized have low
molecular masses (~14 kDa), require millimolar [Ca2+]
for catalytic activity, and function as extracellular secreted enzymes
designated sPLA2 (3, 6). The first PLA2 to be
cloned that is active at [Ca2+] that can be achieved in
the cytosol of living cells is an 85-kDa protein classified as a group
IV PLA2 and designated cPLA2 (3, 5). This
enzyme is induced to associate with its substrates in membranes by
rises in cytosolic [Ca2+] within the range achieved in
cells stimulated by extracellular signals that induce Ca2+
release from intracellular sequestration sites or Ca2+
entry from the extracellular space, is also regulated by
phosphorylation, and prefers substrates with sn-2
arachidonoyl residues (5).
Recently, a second PLA2 that is active at
[Ca2+] that can be achieved in cytosol has been cloned
(8-10). This enzyme does not require Ca2+ for catalysis,
is classified as a group VI PLA2, and is designated iPLA2 (3, 4). The iPLA2 enzymes cloned from
hamster (8), mouse (9), and rat (10) cells represent species homologs, and all are 84-kDa proteins containing 751-752 amino acid residues with highly homologous (~95% identity) sequences. Each
contains a GXSXG lipase consensus motif and eight
stretches of a repeating sequence motif homologous to a repetitive
motif in the integral membrane protein binding domain of ankyrin
(8-10). The substrate preference of these iPLA2 enzymes
varies widely with the mode of presentation (8), but each is
susceptible to inhibition (8-10) by a bromoenol lactone (BEL) suicide
substrate (11, 12) that is not an effective inhibitor of
sPLA2 or cPLA2 enzymes at comparable
concentrations (4, 11-14).
Proposed functions for iPLA2 include a housekeeping
role in phospholipid remodeling that involves generation of
lysophospholipid acceptors for incorporation of arachidonic acid into
phospholipids of murine P388D1 macrophage-like cells (4, 15, 16),
although signaling functions of iPLA2 have been suggested
in other cells (10, 17-21). The proposed housekeeping function for
iPLA2 (4) has been deduced from experiments involving
inhibition of iPLA2 activity in P388D1 cells with BEL (15)
or with an antisense oligonucleotide (16). Inhibition of
iPLA2 activity in P388D1 cells suppresses incorporation of
[3H]arachidonic acid into phospholipids by about 60%,
but [3H]palmitic acid incorporation is reduced only
slightly (15, 16). Incorporation of [3H]arachidonic acid
into P388D1 cells is Ca2+-independent and unaffected by
chelation of extracellular or intracellular Ca2+ (15).
Inhibition of iPLA2 also reduces
[3H]lysophosphatidylcholine (LPC) levels by about 60% in
[3H]choline-labeled P388D1 cells, and this is thought to
represent the mechanism whereby iPLA2 inhibition reduces
incorporation of [3H]arachidonic acid into P388D1 cell
phospholipids (15, 16). Such incorporation (15, 16) reflects a
deacylation/reacylation cycle (22, 23) of phospholipid remodeling
rather than de novo synthesis (24), and the level of LPC
acceptors is thought to limit the rate of [3H]arachidonic
acid incorporation into P388D1 cell phosphatidylcholine (PC) (15, 16).
Activity of iPLA2 appears to be required to maintain
sufficient levels of LPC in P388D1 cells to support
[3H]arachidonic acid incorporation into PC (15, 16).
It is not yet certain that the observations with P388D1 cells reflect a
general mechanism for incorporation of arachidonic acid into
phospholipids of all cells, and P388D1 cells exhibit some atypical
features of arachidonate incorporation. Arachidonate represents about
25% of the total esterified fatty acyl mass in native murine
macrophage phospholipids (25, 26) but only 3% of that in murine P388D1
macrophage-like cells (27-29). A significant portion of exogenous
arachidonate provided to P388D1 cells is also found in the
intracellular free fatty acid fraction (29), and this is not the case
in other cell types (30-32), in which nonesterified arachidonate is
maintained at a very low level by an efficient esterification system
(26, 33-34). In many cells, nonesterified arachidonate imported from
the extracellular space or released intracellularly by PLA2
enzymes is quickly converted to arachidonoyl-CoA in an
ATP-dependent step and then rapidly incorporated into
phospholipids by acyltransferases (35, 36). The low levels of
esterified arachidonate and the relatively high levels of nonesterified
arachidonate observed in P388D1 cells (27-29) suggest that they may be
deficient in arachidonate incorporation mechanisms expressed by other cells.
One biomedically important cell that may require especially effective
arachidonate incorporation mechanisms is the insulin-secreting pancreatic islet beta cell, the function of which is impaired in
diabetes mellitus. Arachidonate represents 30-36% of the total esterified fatty acyl mass in phospholipids of normal rat and human
islets and isolated beta cells (37, 38), and arachidonate-containing species are the most abundant components of all major islet
phospholipid head-group classes (39). The abundance of
arachidonate-containing phospholipid species is higher in islets than
in many other tissues (39), and islet plasma membranes and secretory
granule membranes are especially enriched in such species (38, 39).
Fusion of secretory granule and plasma membranes is the final event in
insulin exocytosis, and the high content of certain
arachidonate-containing phospholipids in those membranes may facilitate
their fusion (39, 40). Because both rat and human beta cells also
express iPLA2 (10, 41), we have examined the participation
of iPLA2 in arachidonic acid incorporation into islet and
insulinoma cell phospholipids, and our findings differ from those in
P388D1 cells.
 |
EXPERIMENTAL PROCEDURES |
Materials--
The compounds
[5,6,8,9,11,12,14,15-3H]arachidonic acid (100 Ci/mmol),
[9,10-3H]palmitic acid (54 Ci/mmol),
[methyl-3H]choline chloride (90 Ci/mmol), and
L-
-dipalmitoyl-[glycerol-U-14C]phosphatidic
acid (200 mCi/mmol) were obtained from NEN Life Science Products. ECL
detection reagents and
1-palmitoyl-2-[14C]linoleoyl-sn-glycero-3-phosphocholine
(55 mCi/mmol) were purchased from Amersham Pharmacia Biotech. The
1-palmitoyl-2-hydroxy-sn-glycerophosphocholine (16:0-LPC),
1-heptadecanoyl-2-hydroxy-sn-glycerophosphocholine (17:0-LPC), and
1-stearoyl-2-hydroxy-sn-glycerophosphocholine (18:0-LPC) and
standard phosphatidylcholine (PC), phosphatidylethanolamine (PE),
phosphatidylserine, and phosphatidic acid (PA) were obtained from
Avanti Polar Lipids (Birmingham, AL).
[5,6,8,9,11,12,14,15-2H8]Arachidonic acid was
prepared by catalytic reduction of eicosa-(5,8,11,14)-tetraynoic acid with 2H2 gas (42). Arachidonic
acid, palmitic acid, triolein, diolein, and monoolein were obtained
from Nu-Chek Prep (Elysian, MN). The bromoenol lactone (BEL)
iPLA2 suicide substrate
(E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one was purchased from Cayman Chemical (Ann Arbor, MI). Male Sprague-Dawley rats (180-220 g) were obtained from Harlan Breeders
(Indianapolis, IN) and collagenase from Roche Molecular Biochemicals.
Tissue culture media (CMRL-1066, RPMI, and minimal essential medium), penicillin, streptomycin, Hanks' balanced salt solution (HBSS), and
L-glutamine were purchased from Life Technologies, Inc.
Fetal bovine serum was obtained from HyClone (Logan, UT), and Pentex bovine serum albumin (BSA, fatty acid free, fraction V) from ICN Biomedicals (Aurora, OH). Rodent Chow 5001 was obtained from Ralston Purina (St. Louis, MO) and D-glucose from the National
Bureau of Standards (Washington, D. C.). ATP, ampicillin, propranolol, and kanamycin were obtained from Sigma. BAPTA-AM and
NG-monomethyl-L-arginine acetate
(NMMA) were obtained from Calbiochem and interleukin-1
(IL-1) from
Cistron Biotechnology (Pine Brook, NJ). Media included KRB
(Krebs-Ringer bicarbonate buffer: 25 mM Hepes, pH 7.4, 115 mM NaCl, 24 mM NaHCO3, 5 mM KCl, 1 mM MgCl2); cCMRL
(CMRL-1066 supplemented with 10% heat-inactivated fetal bovine serum,
1% L-glutamine, 1% penicillin, 1% streptomycin); and
HBSS supplemented with 0.5% penicillin/streptomycin.
Isolation and Culture of Pancreatic Islets and Insulinoma
Cells--
Islets were isolated aseptically from male Sprague-Dawley
rats by collagenase digestion of minced pancreas, density gradient isolation, and manual selection under microscopic visualization (43).
Isolated islets were placed in cCMRL-1066 under a humidified atmosphere
of 95% air, 5% CO2 and cultured at 37 °C. Islet total insulin and protein contents were determined as described previously (44). Islets were isolated from human pancreata in the Core Laboratory
of the Washington University Diabetes Research and Training Center (45)
and were cultured as described (39). INS-1 insulinoma cells provided by
Dr. Christopher Newgard (University of Texas-Southwestern, Dallas, TX)
were cultured under previously described conditions (46-48).
Incubation of Islets and Insulinoma Cells with Radiolabeled Fatty
Acids, BEL, BAPTA, IL-1, and Other Additives--
Islets or INS-1
cells were washed three times in KRB medium containing 5.5 mM glucose and 0.1% BSA, resuspended in that medium, and
preincubated for 30 min at 37 °C. The islets (100 per condition) or
INS-1 cells (2 × 105 per condition) were then placed
in fresh KRB medium that contained glucose (5.5, 11, or 22 mM), 0.1% BSA, and either 2.5 mM
CaCl2 or 1 mM EGTA and no added
Ca2+ and were incubated (30 min, 37 °C) after addition
of vehicle only (control) or BEL (1-25 µM). In
experiments examining iPLA2 activity, control and
BEL-treated islets or INS-1 cells were then either homogenized
immediately or placed in fresh cCMRL medium and incubated at 37 °C
for 8-68 h before homogenization, and iPLA2 activity was
determined in homogenates. In some experiments examining radiolabeled
fatty acid incorporation, effects of loading islets or INS-1 cells with
BAPTA-AM (100 µM, 30 min) or propranolol (250 µM, 30 min) under described conditions (49) before adding
radiolabeled fatty acid were determined. After the preincubation and
loading steps described above, fatty acid incorporation experiments
were initiated by adding either [3H]arachidonic acid
(final concentration 0.5 µCi/ml, 5 nM) or
[3H]palmitic acid (final concentration 0.5 µCi/ml, 10 nM) to the medium, and incubation was performed for 10-60
min at 37 °C. Islets or INS-1 cells were then washed three times in
KRB medium containing 5.5 mM glucose and 0.1% BSA to
remove unincorporated 3H-fatty acid. In some experiments,
cellular lipids were then immediately extracted by the method of Bligh
and Dyer (50) and analyzed by TLC or HPLC. In other experiments, after
initial incubation with [3H]arachidonic acid, islets were
washed to remove unincorporated radiolabel, placed in cCMRL medium,
and incubated (24 h, 37 °C) in the absence of 3H-fatty
acid. The islets were then washed and their lipids extracted and
analyzed. In experiments examining effects of IL-1 on islet fatty acid
incorporation, islets were incubated (24 h, 37 °C) with no
additions, with IL-1 (5 units/ml) alone, or with IL-1 plus NMMA (0.5 mM) under described conditions (51). The islets were then
washed, preincubated, and incubated with 3H-fatty acids as
above. Under all loading and incubation conditions, islet and INS-1
cell viability exceeded 98% by trypan blue exclusion performed as
described (49).
Assay of iPLA2 Activity in Islets and INS-1
Cells--
Control and BEL-treated islets and INS-1 cells were
homogenized by sonication as described (52). Protein content was
measured with Coomassie reagent (Pierce) against bovine serum albumin
as standard. Ca2+-independent PLA2 activity was
assayed by incubating (30 min, 37 °C) aliquots of homogenate in
buffer (100 mM Hepes, pH 7.5, 5 mM EDTA, 400 µM Triton X-100) with 100 µM
1-palmitoyl-2-[14C]linoleoyl-sn-glycero-3-phosphocholine
in a final volume of 500 µl with or without 1 mM ATP. The
substrate was presented in mixed micelles of Triton X-100/phospholipid
at a molar ratio of 4/1, obtained by a combination of heating, vortex
mixing, and bath sonication. Products were analyzed by TLC as described
below, and PLA2 specific activity was calculated from
disintegrations/min of released [14C]linoleate and
protein content as described (52).
Chromatographic Analyses of Radiolabeled
Phospholipids--
Incorporation of 3H-fatty acids into
islet and INS-1 cell phospholipids was determined by TLC (15, 16) or by
HPLC (37, 49, 53). TLC was performed on silica gel G plates with
hexane/diethyl ether/acetic acid (70/30/1). Phospholipids remain at the
origin and are resolved from diglycerides (Rf
0.21-0.24), free fatty acids (Rf 0.58), and
triglycerides. Phospholipid head-group classes were separated by
NP-HPLC (37, 49, 53) analyses on a silicic acid column (LiChrosphere
Si-100, 10 µm, 250 × 4.5 mm, Alltech, Deerfield, IL) with the
solvent system hexane/2-propanol/(25 mM potassium
phosphate, pH 7.0)/ethanol/acetic acid (367/490/62/100/0.6) at a flow
of 0.5 ml/min for 60 min and then 1.0 ml/min. Retention times of
standard phospholipids were as follows: PE, 11 min; PA, 20 min; PI, 27 min; phosphatidylserine 38 min; and PC, 102 min. Molecular species of
PC were separated by RP-HPLC (Ultrasphere ODS column, 4.6 × 250 mm, 5-µm particle size, Alltech, Deerfield, IL) with 20 mM choline chloride in methanol/water/acetonitrile (90.5/7/2.5) at a flow of 2.0 ml/min at 40 °C (37, 53, 54). Retention times for standard 16:0/20:4-GPC and 18:0/20:4-GPC were 31 and 48 min, respectively.
Immunoblotting Analyses of INS-1 Cell iPLA2
Protein--
INS-1 cell cytosolic proteins were analyzed by SDS-PAGE
and transferred to a nylon membrane that was subsequently blocked (3 h,
room temperature) with Tris-buffered saline/Tween (TBS-T, which
contains 20 mM Tris-HCl, 137 mM NaCl, pH 7.6, and 0.05% Tween 20) containing 5% milk protein. The blot was then
washed (TBS-T, 5 min, five times) and incubated (1 h, room temperature) with a polyclonal antibody (1:4000 dilution in TBS-T) to
iPLA2 provided by Dr. Simon Jones (Genetics Institute,
Boston). The nylon membrane was then washed in TBS-T (5 min, five
times) and incubated (1 h, room temperature) with a secondary antibody
coupled to horseradish peroxidase (Roche Molecular Biochemicals) at
1:20,000 dilution in TBS-T containing 3% BSA. The antibody complex was visualized by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech).
Isolation of RNA from INS-1 Cells, Reverse Transcription, and
Polymerase Chain Reaction Amplification of an iPLA2
cDNA Fragment--
Total RNA was isolated from INS-1 cells and
first strand cDNA prepared by reverse transcription (RT) using
standard methods (8, 55). Polymerase chain reactions (PCR) were
performed and products analyzed as described elsewhere (8). Primers
used to amplify a rat iPLA2 cDNA fragment were sense
(5'-TGCAGACCAGTTAGTATGGC-3') and antisense (5'-TGGACAAGCTTCTGGAAC-3').
The expected fragment length is 528 bp, and digestion with restriction
endonuclease SmaI is expected to yield 164- and 364-bp fragments.
Attempts at Suppressing INS-1 Cell iPLA2 Expression
with Antisense Oligonucleotides--
Many attempts to suppress INS-1
cell iPLA2 expression with antisense oligonucleotides were
performed using different oligonucleotide sequences at various
concentrations, durations of exposure, and presentation conditions. The
first mimicked that applied to P388D1 cells (16) and employed a 20 base-long antisense oligonucleotide corresponding to nucleotides 59-78
in the rat group VI iPLA2 sequence (10). The
oligonucleotide sequence is 5'-CTCCTTTGCCCGGAATG-GGT-3', and a sense
oligonucleotide was used as control. Both contained phosphorothioate
linkages to limit degradation. For transfection, 2.5 × 105 cells were cultured with oligonucleotide in Iscove's
modified Dulbecco's medium for 4 h in a humidified atmosphere at
90% air and 10% CO2. A final concentration of 10% fetal
bovine serum was then added, and the cells were cultured for an
additional 48 h in medium supplemented with 2 mM
glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and
nonessential amino acids. At oligonucleotide concentrations from 0.10 to 20 µM, there was no suppression of INS-1 cell
iPLA2 protein or activity, and cell detachment occurred at
higher concentrations, as for P388D1 cells (16). Oligonucleotide presentation in various concentrations of Lipofectin (56) to cells
attached to surfaces and in suspension was also examined, as were two
other antisense oligonucleotide sequences. One
(5'-AGGCGTCCAAAG-AACTGC-3') was designed hybridize to the mRNA
region containing the initiator codon and the other
(5'-TTGTTGTCATGTACATCC-3') to a region containing a methionine codon
upstream of the catalytic center coding region, but neither was effective.
Phosphatidate Phosphohydrolase Assays--
These assays examined
conversion of [glycerol-U-14C])phosphatidic
acid (PA) to [14C]diacylglycerol catalyzed by
phosphatidate phosphohydrolase (PAPH) (57-59) in islet cytosol and
membranes. Islets (4000 per experiment) were washed twice with
homogenization buffer (50 mM Tris, pH 6.5, 1 mM
EDTA, 1 mM EGTA), resuspended in that buffer (0.3 ml),
disrupted by sonication (Vibra Cell sonicator, 8 s, power setting
12), and centrifuged (45 min, 4 °C, 14,000 × g).
The cytosolic supernatant was removed and the membranous pellet
resuspended in homogenization buffer, and their protein contents were
determined. Substrate [14C]PA was presented in mixed
micelles with Triton X-100 at a detergent phospholipid ratio of 10/1.
The incubation mixture contained (final volume 0.1 ml) 100 µM [14C]PA (0.025 µCi/assay), 1 mM Triton X-100, 50 mM Tris-HCl, pH 6.5, 10 mM
-mercaptoethanol, 3 mM MgCl2,
1 mM EGTA, 1 mM EDTA, and 100 µg of cytosolic
or 50 µg of membranous protein. MgCl2 was omitted from
some reactions. When NEM (8 mM) or BEL (25 µM) was used, samples were incubated with the reagents
for 10 min before adding [14C]PA. Assays were conducted
at 37 °C for 30 min. Reactions were terminated by extraction with 2 ml of chloroform/methanol (1/1). The chloroform layer was concentrated
and analyzed by TLC on silica gel G plates with toluene/ethyl
acetate/diethyl ether/acetic acid (80/10/10/0.2). [14C]PA
remains at the origin, and the PAPH reaction product
[14C]diacylglycerol (Rf 0.60) is
separated from monoacylglycerol (Rf 0.13),
triacylglycerol (Rf 0.89), and free fatty acid
(Rf 0.33). The 14C content of the
diacylglycerol region was quantified by liquid scintillation
spectrometry. Assays were linear with time and protein concentration
and exhibited zero order kinetics under these conditions.
Islet de Novo Synthesis of Glycerolipids from
[14C]Glucose--
Islet synthesis of glycerolipids from
[14C]glucose was examined by a modification of described
methods (60). Islets suspended in KRB medium containing 5.5 mM glucose and 0.1% BSA were placed (100/condition) in
silanized test tubes and preincubated (30 min, 37 °C) with vehicle
only (control) or with BEL (25 µM).
[14C]Glucose (final concentration 20 µCi/ml) was then
added, and the islets were incubated for 60 min at 37 °C. Medium was
then removed and the islets washed with KRB medium containing 5.5 mM glucose. Islet lipids were then extracted (50), and the
extract was concentrated and analyzed by TLC on silica gel G plates
with toluene/diethyl ether/ethyl acetate/acetic acid (80/10/10/0.2). Phospholipids, diacylglycerol, and triacylglycerol were recovered from
the plate and their [14C] contents determined by liquid
scintillation spectrometry.
Incubation of INS-1 Cells with Arachidonic Acid to Induce
Phospholipid Remodeling--
INS-1 insulinoma cells cultured in RPMI
medium supplemented with penicillin, streptomycin, fungizone, and
gentamicin at a concentration of 0.1% (w/v) each. Cells (1.2 × 106 per condition) were treated (30 min, 37 °C) with
vehicle only or with BEL (25 µM). Medium was then removed
and replaced with fresh medium containing no supplements other than
those described above or containing arachidonic acid (final
concentration 70 µM), and cells were cultured at 37 °C
for various periods. After 0, 8, or 24 h, cells were washed twice
with phosphate-buffered saline, suspended in homogenization buffer, and
disrupted by sonication. One aliquot of homogenate was used to measure
protein content and iPLA2 activity, and the remainder was
extracted by the method of Bligh and Dyer (50). The extract was
concentrated and analyzed by NP-HPLC to separate phospholipid
head-group classes. GPC lipids were analyzed by ESI/MS.
Effects of BEL on Islet Lysophosphatidylcholine (LPC) Mass and on
the [3H]LPC Content of Islets Labeled with
[3H]Choline--
To determine islet LPC mass, islets
(1500 per condition) were washed twice with and then resuspended and
incubated (30 min, 37 °C) in KRB medium containing 5.5 mM glucose and 0.1% BSA. Islets were then placed in fresh
medium and incubated (30 min, 37 °C) with vehicle only (control) or
with BEL (25 µM). To one set of islets, bee venom
sPLA2 (50 units/ml, Sigma) and CaCl2 (2.5 mM) were added, and incubation was continued (30 min,
37 °C). Islets were then washed twice with phosphate-buffered saline
and extracted by the method of Bligh and Dyer (50) with the
modifications that 150 mM LiCl in water was aqueous phase
and that the chloroform phase contained 17:0-LPC internal standard (300 pmol per condition). Concentrated extracts were analyzed by silica gel
G TLC on heat-activated (30 min, 80 °C) plates with
chloroform/methanol/ammonium hydroxide (65/30/0.8) to separate LPC
(Rf 0.38), LPE (Rf 0.46), and PC
(Rf 0.60). LPC was extracted by the method of Bligh
and Dyer (50) with the modification that 150 mM LiCl in
water was aqueous phase and analyzed by ESI/MS/MS.
[3H]LPC content of [3H]choline-labeled
islets was examined by a modification of described methods (61). Islets
were cultured (40 h, 37 °C) in cCMRL medium containing
[3H]choline chloride (20 µCi/ml) and then washed with
and resuspended (300 islets/condition) in KRB medium containing 5.5 mM glucose and 0.1% BSA and incubated (30 min, 37 °C).
Medium was removed and replaced with fresh medium supplemented with
vehicle only (control) or with BEL (25 µM), and
incubation was performed (30 min, 37 °C). Medium was removed and
replaced with fresh medium, and incubation was performed (10 min,
37 °C). Islets were washed with KRB containing 5.5 mM
glucose and 0.1% BSA and extracted (50). Concentrated extracts were
analyzed by TLC to isolate LPC, and its 3H content was determined.
Electrospray Ionization Mass Spectrometric Analyses of
Choline-containing Lipids--
PC and LPC were analyzed as
Li+ adducts by ESI/MS on a Finnigan (San Jose, CA) TSQ-7000
triple stage quadrupole mass spectrometer with an ESI source controlled
by Finnigan ICIS software. Phospholipids were dissolved in
methanol/chloroform (9/1, v/v) containing LiOH (2 nmol/µl), infused
(1 µl/min) with a Harvard syringe pump, and analyzed under described
instrumental conditions (39, 49, 62). For tandem MS, precursor ions
selected in the first quadrupole were accelerated (32-36 eV collision
energy) into a chamber containing argon (2.3-2.5 millitorr) to induce
collisionally activated dissociation, and product ions were analyzed in
the final quadrupole. Identities of GPC species in the total ion
current profile were determined from their tandem spectra (62). To
quantitate LPC species, constant neutral loss scanning was performed to
monitor LPC [M + Li]+ ions that undergo loss of 59 (trimethylamine) upon collisionally activated dissociation.
Quantitation was achieved by comparing the intensity of the ion for the
17:0-LPC internal standard (m/z 516) to intensities of ions
for the endogenous islet LPC species (63) 16:0-LPC (m/z 502)
and 18:0-LPC (m/z 530). Standard curve experiments in which
a constant amount of standard 17:0-LPC and varied amounts of standard
16:0-LPC and 18:0-LPC were added to a series of tubes and analyzed as
Li+ adducts by ESI/MS/MS indicated that this method was
linear over a wide range that included levels observed in islets.
Examination of Insulin Secretagogue-induced Hydrolysis of
Arachidonic Acid from Islet Phospholipids by Isotope Dilution Gas
Chromatography-Negative Ion Electron Capture-Mass
Spectrometry--
Accumulation of nonesterified arachidonic acid in
islet incubations was determined by modifications of described methods
(42, 49). Islets (300 per condition) were incubated (30 min, 37 °C) in KRB medium containing 3 mM glucose, 2.5 mM
CaCl2, 0.1% BSA, and vehicle only (control), BEL (25 µM), or propranolol (250 µM). Medium was
then removed and replaced with fresh medium. To some tubes no
supplements were added, and to others either supplemental glucose
(final concentration 17 mM) or glucose plus carbachol (final concentration 0.5 mM) was added. Incubation was then
performed (30 min, 37 °C) and terminated by extraction with 2 ml of
chloroform/methanol (1/1) containing 825 pmol of
[2H8]arachidonic acid internal standard.
Extracts were analyzed by RP-HPLC to isolate arachidonic acid, which
was converted to a pentafluorobenzyl ester derivative and analyzed by
GC/MS in negative ion electron capture mode under described conditions (42, 49). Selected monitoring of carboxylate anions of arachidonate (m/z 303) and [2H8]arachidonate
(m/z 311) was performed to quantitate arachidonate by
reference to a standard curve (42, 49).
Statistical Analyses--
Student's t test was used
to compare two groups, and multiple groups were compared by one-way
analysis of variance with post hoc Newman-Keul's analyses.
 |
RESULTS |
In the report that motivated our study, 25 µM BEL
maximally inhibited iPLA2 activity and suppressed
[3H]arachidonic acid incorporation into P388D1 cell
phospholipids during a 10-min incubation but only slightly suppressed
[3H]palmitic acid incorporation, and
[3H]arachidonic acid incorporation was unaffected by
chelation of extracellular or intracellular [Ca2+] (15).
To examine the generality of these phenomena, we performed similar
experiments with islets. Control islets exhibited PLA2 activity in the absence of Ca2+ that was stimulated by ATP,
and activity was 98% inhibited in 25 µM BEL-treated
islets (Fig. 1, left panel),
consistent with properties of recombinant iPLA2 expressed
from cDNA cloned from an islet library (10). Despite effective
iPLA2 inhibition, no suppression of
[3H]arachidonic acid incorporation into phospholipids was
observed in BEL-treated islets during 10-min incubations in
Ca2+-replete medium, in Ca2+-free
EGTA-containing medium, or in islets that had been loaded with the
intracellular Ca2+-chelator BAPTA-AM and incubated in
Ca2+-free EGTA-containing medium (Fig. 1, center
panel). [3H]Arachidonic acid incorporation was
greater in Ca2+-free EGTA-containing medium than in
Ca2+-replete medium, consistent with reports that exogenous
arachidonate achieves greater intracellular access in islets (64, 65)
and other tissues (66) in Ca2+-free compared with
Ca2+-replete medium and that this also occurs with
palmitate (67).

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Fig. 1.
Effects of the iPLA2
suicide substrate BEL on pancreatic islet
iPLA2 activity and on incorporation of
[3H]arachidonic acid and of
[3H]palmitic acid into islet
phospholipids. Islets were exposed to vehicle
(cross-hatched bars) or 25 µM BEL (dark
bars) for 30 min at 37 °C and were then homogenized and assayed
for iPLA2 activity (left panel) or incubated
with [3H]arachidonic acid (center panel) or
[3H]palmitic acid (right panel) for 10 min at
37 °C. Assays for iPLA2 activity were conducted in
Ca2+-free, 1 mM EGTA-containing medium without
(EGTA ONLY) or with 1 mM ATP
(ATP+EGTA). Incorporation of 3H-fatty acids into
islet phospholipids was determined in medium containing 2.5 mM CaCl2 (CALCIUM), medium
containing 1 mM EGTA and no added Ca2+
(EGTA), or with BAPTA-loaded islets incubated in
Ca2+-free, EGTA containing medium (BAPTA). At
the end of incubations, islet lipids were extracted and analyzed by TLC
to isolate phospholipids. Results are expressed as 3H
disintegrations/min in phospholipids per 20 µg of islet protein
(n = 27). The p values for the differences
in incorporation of 3H-fatty acids in control and
BEL-treated islets were less than 0.05 for all relevant comparisons
except for the BAPTA condition in the center panel. The
p values for differences in incorporation of
3H-fatty acids in the CALCIUM, EGTA,
and BAPTA conditions were less than 0.05 for all relevant
comparisons.
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|
In Ca2+-free EGTA-containing medium, non-BAPTA-loaded
islets incorporated more [3H]arachidonic acid into
phospholipids than did BAPTA-loaded islets (Fig. 1, center
panel), under conditions where BAPTA reduces islet beta cell
cytosolic [Ca2+] by 50% and prevents ionophore
A23187-induced rises in cytosolic [Ca2+] (49). This
indicates that [3H]arachidonic acid incorporation into
islet phospholipids is not completely Ca2+-independent.
Incorporation of [3H]palmitic acid into islet
phospholipids (Fig. 1, right panel) exhibited features
similar to those for [3H]arachidonic acid, and
incorporation of both fatty acids was generally higher in BEL-treated
than control islets. This effect was observed at all incubation times
between 10 and 60 min (Fig. 2), and no
suppression of [3H]arachidonic acid incorporation into
islet phospholipids was observed at any BEL concentration that reduced
islet iPLA2 activity (Fig.
3). Incorporation of both fatty acids
into human islet phospholipids was affected by BEL and by manipulating
Ca2+ in ways similar to those with rat islets (not
shown).

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Fig. 2.
Time course of incorporation of
[3]arachidonic acid or [3]palmitic acid
into islet phospholipids. Islets were incubated with vehicle
(open symbols) or 25 µM BEL (closed
symbols) for 30 min at 37 °C and were then incubated with
[3H]arachidonic acid (left panel) or
[3H]palmitic acid (right panel) for 10-60
min. The 3H content of islet phospholipids was determined
as in Fig. 1 and is expressed as disintegrations/min per 20 µg of
islet protein (n = 6).
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Fig. 3.
Effects of varied concentrations of BEL on
islet iPLA2 activity and on incorporation of
[3]arachidonic acid into islet phospholipids.
Islets were incubated with vehicle or with 1, 3, or 10 µM
BEL for 30 min at 37 °C. Islets were then homogenized and assayed
for iPLA2 activity (left panel) or incubated
with [3H]arachidonic acid for 10 min at 37 °C
(right panel). Incorporation of
[3H]arachidonic acid into islet phospholipids was
determined as in Fig. 1 and is expressed as disintegrations/min per 20 µg of islet protein (n = 9).
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|
To determine whether it is possible to suppress fatty acid
incorporation into islet phospholipids under our conditions, we examined effects of incubating islets with interleukin-1 (IL-1) (Fig.
4), which stimulates islet production of
nitric oxide and causes accumulation of nonesterified arachidonic acid
by an NO-dependent mechanism that is thought to reflect
impaired arachidonoyl-CoA generation (51). Incubating islets with IL-1
impairs incorporation of [3H]arachidonic acid into islet
phospholipids in Ca2+-replete medium (51), and we found
that this is also true in Ca2+-free medium and that
incorporation of [3H]palmitic acid is similarly
suppressed (Fig. 4). Coincubation of islets with IL-1 and the nitric
oxide synthase inhibitor NMMA prevented IL-1-induced suppression of
fatty acid incorporation into islet phospholipids (Fig. 4), reflecting
the NO dependence of this effect. Fatty acid incorporation into islet
phospholipids thus can be suppressed, even though inhibiting islet
iPLA2 activity does not do so.

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Fig. 4.
Suppression of [3]arachidonic
acid and of [3]palmitic incorporation into islet
phospholipids after incubation with interleukin-1 and prevention of
this effect by a nitric oxide synthase inhibitor. Islets
were incubated with vehicle (CONTROL), with 5 units/ml
interleukin-1 alone (IL-1), or with IL-1 plus 0.5 mM NMMA (IL-1+NMMA) for 24 h at 37 °C.
Islets were then incubated with [3H]arachidonic acid
(dark bars) or with [3H]palmitic acid
(cross-hatched bars) for 10 min at 37 °C in medium
containing 1 mM EGTA and no added Ca2+. The
3H content of islet phospholipids was then determined as in
Fig. 1 and is expressed as disintegrations/min per 20 µg of islet
protein (n = 6).
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|
To attempt to inhibit beta cell iPLA2 activity with
antisense oligonucleotides, we examined INS-1 insulinoma cells because islets are a multicellular aggregate in which it is difficult to
achieve effective intracellular levels of oligonucleotide. INS-1 cells
are derived from rat islet beta cells (46-48) and were found to
express a PLA2 activity in the absence of Ca2+
that is stimulated by ATP and inhibited by BEL (Fig.
5A), an 84-kDa protein
recognized by an iPLA2 antibody (Fig. 5B), and an mRNA species that is amplified in RT-PCR reactions with rat iPLA2-specific primers and that contains the expected
SmaI restriction endonuclease cleavage site (Fig.
5C). We were unable to suppress INS-1 cell iPLA2
activity or protein with an antisense oligonucleotide corresponding in
sequence, except for minor differences between species, to one that
suppresses P388D1 cell iPLA2 expression (16). Two other
antisense oligonucleotide sequences presented under a variety of
conditions were also ineffective.

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Fig. 5.
Expression of iPLA2 by INS-1
insulinoma cells. A, homogenates of INS-1 cells
were assayed for iPLA2 activity in medium containing 1 mM EGTA and no added Ca2+ without BEL or ATP
(EGTA) or in medium supplemented either with 10 µM BEL (EGTA+BEL) or with 1 mM ATP
(EGTA+ATP). B, aliquots of INS-1 cell cytosol
containing 1 µg (lane 1), 10 µg (lane 2), or
20 µg (lane 3) of protein were analyzed by SDS-PAGE, and
immunoblotting was performed with an antibody against iPLA2
provided by Dr. Simon Jones. C, RNA was prepared from INS-1
cells and used as template in RT-PCR reactions with rat
iPLA2-specific primers that were expected to yield a
product of 528 bp in length, based on the rat iPLA2
cDNA sequence (10). RT-PCR products were analyzed by agarose gel
electrophoresis and visualized with ethidium bromide. Lane 1 represents a control reaction performed without an RT step to exclude
contamination of the RNA with genomic DNA. Lanes 2 and
3 represent reaction products obtained from RT-PCR reactions
performed with two separate preparations of INS-1 cell RNA. Lanes
4 and 5 represent products obtained from digestion of
the DNA species visualized in lanes 2 and 3 with
the restriction endonuclease SmaI, which was expected to
yield products of 164 and 364 bp lengths, based on the rat
iPLA2 cDNA sequence (10).
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|
Nonetheless, because INS-1 cell iPLA2 activity is inhibited
by BEL, we compared effects of BEL on fatty acid incorporation into
phospholipids of islets and INS-1 cells. We also examined effects of
propranolol because both BEL and propranolol inhibit phosphatidate
phosphohydrolase (PAPH) in some cells (57, 70). At 250 µM, propranolol maximally inhibits PAPH in islets (71) and amniotic WISH cells (70) and suppressed islet de novo
synthesis of triacylglycerol, as do PAPH inhibitors in P388D1 cells
(29, 57). BEL did not suppress incorporation of
[3H]arachidonic acid or [3H]palmitic
acid into islet (Fig. 6, A and
B) or INS-1 cell phospholipids (Fig. 6, C and
D) in Ca2+-replete or Ca2+-free
EGTA-containing medium, and incorporation of neither fatty acid was
significantly affected by propranolol in islets or INS-1 cells (Fig.
6).

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Fig. 6.
Comparisons of effects of BEL and of the
phosphatidate phosphohydrolase inhibitor propranolol on incorporation
of [3]arachidonic acid and [3]palmitic acid
into phospholipids of islets and INS-1 insulinoma cells.
Islets (A and B) or INS-1 cells (C and
D) were incubated with vehicle (cross-hatched
bars), 250 µM propranolol (stippled
bars), or BEL (dark bars) for 30 min at 37 °C and
were then incubated with [3H]arachidonic acid
(A and C) or with [3H]palmitic acid
(B and D) for 10 min at 37 °C in medium
containing 2.5 mM CaCl2 (CALCIUM) or
1 mM EGTA and no added Ca2+ (EGTA).
Propranolol (250 µM) was included in the incubation
medium of the islets or INS-1 cells that had been preincubated with
that agent. Incorporation of 3H-fatty acids into
phospholipids was determined as in Fig. 1 and is expressed as
disintegrations/min per 20 µg of islet protein or per 200 µg of
INS-1 cell protein (n = 12). The p values
for differences between 3H incorporation into control and
BEL-treated cells were less than 0.05 for all relevant comparisons and
were greater than 0.05 for differences between control and
propranolol-treated cells for all such comparisons.
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|
One possible explanation for the failure of BEL to suppress fatty acid
incorporation into islet phospholipids is that, by inhibiting PAPH, BEL
causes accumulation of phosphatidic acid (PA) and enhances de
novo phosphatidylinositol (PI) synthesis (24, 72). This might mask
suppression of [3H]arachidonic acid incorporation into
GPC lipids by deacylation/reacylation. We first determined whether BEL
inhibits islet PAPH activity. PAPH isoforms include PAPH-1, which is
Mg2+-dependent and inhibited by NEM (58, 73)
and BEL, and PAPH-2, which is Mg2+-independent,
NEM-insensitive, and resistant to BEL (57). Islets were found to
express Mg2+-dependent, NEM-sensitive PAPH
activity both in cytosol and membranes (Table
I), consistent with a previous report
(74). BEL (25 µM) reduced islet cytosolic
Mg2+-dependent, NEM-sensitive PAPH activity by
48% but had no effect on membranous
Mg2+-dependent, NEM-sensitive PAPH activity
(Table I). Neither BEL nor NEM affected islet PAPH activity in the
absence of Mg2+, and the combination of BEL and NEM
achieved no greater inhibition of cytosolic PAPH activity than NEM
alone (not shown).
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Table I
Inhibition of phosphatidate phosphohydrolase activity in pancreatic
islet cytosol and membranes by NEM and by BEL
Cytosol and membranes were prepared from homogenized pancreatic islets
and assayed for phosphatidate phosphohydrolase activity in medium
containing 3 mM MgCl2 by following conversion of
[glycerol-U-14C]phosphatidic acid to
[14C]diacylglycerol, and effects of treating islet cytosol
with 8 mM NEM or 25 µM BEL on the conversion
were determined (n = 12). The p values for
the differences between the control condition and the NEM and BEL
conditions were each less than 0.05, as was that between the NEM and
BEL conditions.
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The inhibition of islet cytosolic PAPH-1 activity by BEL caused us to
examine the possibilities that BEL stimulates incorporation of fatty
acids into PA and PI via de novo synthesis while it reduces incorporation into phosphatidylcholine (PC) by deacylation/reacylation. Islet phospholipids into which [3H]arachidonic acid is
incorporated were analyzed by NP-HPLC (Fig. 7). After 10 min of incubation with
[3H]arachidonic acid, the majority of [3H]
was incorporated into PC in both control, as previously observed (63),
and BEL-treated islets. BEL-treated islets exhibited greater [3H]arachidonic acid incorporation into PC than control
islets, and the profile of [3H]arachidonic acid-labeled
phospholipid head-group classes was similar in the two groups (Fig.
7A). Upon removal of exogenous [3H]arachidonic
acid and continued culture, a time-dependent decline in the
[3H]arachidonate content of PC and a rise in that of
phosphatidylethanolamine (PE) occurred, as previously observed in
islets (63) and other cells (34), and these effects occurred in both
BEL-treated and control islets (Fig. 7B). RP-HPLC analyses
of PC molecular species labeled with [3H]arachidonic acid
were also similar in control and BEL-treated islets (Fig.
7C). PI contained only a minor fraction of
[3H]arachidonic acid incorporated into phospholipids in
BEL-treated or control islets. These findings indicate that
accumulation of PA and PI does not mask a suppression by BEL of
[3H]arachidonate incorporation into islet PC and PE and
that BEL has little effect on the phospholipid head-group classes into which [3H]arachidonate is initially incorporated or on
its subsequent transfer from PC to other lipids.

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Fig. 7.
Chromatographic analyses of pancreatic islet
phospholipids into which [3]arachidonic acid is
incorporated. Islets were treated with vehicle (open
symbols) or 25 µM BEL (closed symbols)
for 30 min at 37 °C and were then incubated with
[3H]arachidonic acid for 10 min at 37 °C.
A, phospholipids were then immediately extracted and
analyzed by NP-HPLC to separate phospholipid head-group classes.
B, after the initial 10-min labeling period, the medium was
removed and replaced with fresh medium that did not contain
[3H]arachidonic acid, and the islets were incubated for
an additional 24 h. Phospholipids were then extracted and analyzed
by NP-HPLC. NL denotes neutral lipids; PE,
phosphatidylethanolamine; PA, phosphatidic acid;
PI, phosphatidylinositol; and PC,
phosphatidylcholine. C, the PC peak was collected and then
analyzed by RP-HPLC to separate PC molecular species. The fatty acid
substituents of the observed species are indicated.
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To examine further any contribution of de novo phospholipid
synthesis (24) to [3H]arachidonic acid incorporation into
islet phospholipids, effects of medium glucose concentration were
determined (Table II, top). De
novo phospholipid synthesis from glucose requires glycolytic metabolism to triose phosphates and acylation of glycerol 3-phosphate and then lysophosphatidic acid to yield PA (24). Kinetic properties of
the predominant islet glucose transporter and glucokinase cause islet
glycolytic flux to rise severalfold as the medium glucose concentration
increases from 5 to 20 mM (75), and this promotes islet
de novo glycerolipid synthesis (60). Incorporation of [3H]arachidonic acid into phospholipids was found not to
be significantly affected by medium glucose concentration in control or
BEL-treated islets, although the latter exhibited greater incorporation
at each glucose concentration (Table II, top). This indicates that de novo synthesis is not a major contributor to
incorporation of [3H]arachidonic acid into islet
phospholipids under our conditions and cannot account for effects of
BEL on incorporation. This is supported by the finding that conversion
of [14C]glucose to [14C]glycerolipids is
reduced rather than increased in BEL-treated islets (Table II,
bottom).
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Table II
Effects of medium glucose concentration on incorporation of
[3H]arachidonic acid into islet phospholipids and of BEL on
islet de novo synthesis of glycerolipids from
[14C]glucose
Islets were treated with vehicle (Control) or 25 µM BEL
for 30 min at 37 °C and then incubated with
[3H]arachidonic acid (top) in medium containing 5.5, 11, or
22 mM glucose or were incubated with [14C]glucose
(bottom) in medium containing a total glucose concentration of 5.5 mM for 10 min at 37 °C. At the end of the incubation
with the radiolabeled precursor, islet lipids were extracted and
analyzed by TLC. Top, incorporation of [3H]arachidonic acid
into phospholipids is expressed as [3H] dpm per 20 µg of islet protein (n = 6). The p values
for the differences between control and BEL-treated islets were less
than 0.05 at all glucose concentrations, and the values for differences
among glucose concentrations for either control or BEL-treated islets
were greater than 0.05 for all relevant comparisons. Bottom,
incorporation of [14C]glucose into islet glycerolipid classes
is expressed as [14C] dpm per 20 µg of islet protein
(n = 6). The p values for the differences
between control and BEL-treated islets were less than 0.05 for all
glycerolipid classes.
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Recovery of iPLA2 activity in BEL-treated islets occurs
slowly and substantial inhibition persists for many hours (51), raising
the possibility that longer term arachidonate incorporation studies can
be performed without repeated manipulation of the cells. When islets
were exposed to 25 µM BEL and then cultured for various
periods, 99, 92, and 80% inhibition of iPLA2 activity was
observed at 0, 8, and 24 h after treatment, respectively (Fig. 8). Mean iPLA2 activity for
the entire 24-h period following BEL treatment was about 10% that in
control islets. Similar experiments with INS-1 cells indicated that
iPLA2 activity was inhibited by 99, 92, and 60% at 0, 8, and 24 h, respectively, after treatment and that the mean
iPLA2 activity in the 24-h period following BEL treatment
was about 20% of control values (Fig. 8).

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Fig. 8.
Time course for recovery of iPLA2
activity after treatment of islets or INS-1 insulinoma cells with
BEL. Islets (circles) or INS-1 cells
(triangles) were treated with vehicle or with 25 µM BEL for 30 min at 37 °C and then placed in fresh
medium without BEL and incubated for 8-68 h. At various times after
initial exposure to BEL, islets or INS-1 cells were homogenized, and
iPLA2 activity was determined. Activity values are
expressed as the specific activity in the BEL-treated preparations
divided by that in the non-BEL-treated preparations (n = 8).
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Similar experiments were performed to examine effects of
iPLA2 inhibition on incorporation of arachidonate mass into
INS-1 cell phospholipids to complement the radiochemical data.
Electrospray ionization (ESI) mass spectrometric (MS) analyses of
control INS-1 cell glycerophosphocholine (GPC) lipids as
Li+ adducts indicated that arachidonate-containing species
are not abundant in unsupplemented cells but that their abundance
increases substantially within hours after adding arachidonic acid to
the medium (Fig. 9). Fig. 9A
is the ESI/MS total positive ion current of GPC-Li+ lipid
species from control INS-1 cells. Tandem MS analyses (62) established
that the major ions in this profile represent 16:0/16:1-GPC (m/z 738), 16:1/18:1-GPC (m/z 764), 16:0/18:1-GPC
(m/z 766), 18:1/18:1-GPC (m/z 792), and
18:0/18:1-GPC (m/z 794). Arachidonate-containing GPC species
at m/z 788 (16:0/20:4-GPC), m/z 814 (18:1/20:4-GPC), and m/z 816 (18:0/20:4-GPC) are not
abundant. Fig. 9B illustrates that, when INS-1 cells are
incubated with arachidonic acid, by 8 h there is substantial
accumulation of 16:0/20:4-GPC (m/z 788). Fig. 9C
illustrates that at 24 h the abundance of this species increases
further and that 18:0/20:4-GPC (m/z 816) also becomes abundant, a sequence consistent with reports that 16:0/20:4-GPC is a
primary remodeling product and that 18:0/20:4-GPC is a secondary product from remodeling at both sn-1 and sn-2
positions (76-80). Remodeling of INS-1 cell phospholipids with
arachidonate was not suppressed by BEL treatment and was accelerated at
8 h after treatment (Fig. 10),
despite the fact INS-1 cell iPLA2 activity was inhibited by
an average of about 96% during the first 8 h after treatment (Fig. 8).

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Fig. 9.
Electrospray ionization/mass spectrometric
analysis of INS-1 cell glycerophosphocholine lipid species. INS-1
cells were treated with vehicle (A-C) or BEL (D)
for 30 min at 37 °C and then incubated without (A) or
with (B-D) exogenous, unlabeled arachidonic acid (70 µM) for 8 h (A and B) or
24 h (C and D). At the end of the
incubation, lipids were extracted and analyzed by NP-HPLC to isolate
GPC lipids, which were then analyzed as Li+ adducts by
positive ion ESI/MS. Total ion current tracings are displayed.
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Fig. 10.
Time course of appearance of
arachidonate-containing glycerophosphocholine lipid molecular species
in INS-1 cells incubated without or with exogenous arachidonic
acid. INS-1 cells were incubated with vehicle
(triangles and open circles) or 25 µM BEL (closed circles) for 30 min at 37 °C
and then incubated without (triangles) or with
(circles) 70 µM arachidonic acid for 0, 8, or
24 h. Lipids were then extracted and analyzed by NP-HPLC to
isolate GPC lipids, which were analyzed as Li+ adducts by
positive ion ESI/MS. The fraction of the total ion current represented
by the arachidonate-containing species 16:0/20:4-GPC (m/z
788), 18:1/20:04-GPC (m/z 814), and 18:0/20:4-GPC
(m/z 816) was then determined for each condition
(n = 6).
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Suppression of arachidonate incorporation by iPLA2
inhibition in P388D1 cells is proposed to reflect reduction in
lysophosphatidylcholine (LPC) acceptor molecules, the abundance of
which is thought to limit the rate of arachidonate incorporation and to
be governed by iPLA2 activity (15, 16). To determine
effects of iPLA2 inhibition on islet LPC mass, ESI tandem
MS experiments were performed (Fig.
11). After incubation of islets with
BEL or vehicle, internal standard 17:0-LPC, which does not occur
naturally in islets, was added to islet lipid extracts, and LPC species
were isolated by TLC and analyzed as Li+ adducts by
ESI/MS/MS scanning for neutral loss of trimethylamine, an approach that
yields a better signal to noise ratio than does monitoring the total
ion current. Comparing relative intensities of ions for
17:0-LPC-Li+ (m/z 516) and
16:0-LPC-Li+ (m/z 502) or
18:0-LPC-Li+ (m/z 530) yields a linear standard
curve over a wide concentration range that includes LPC levels observed
in islets.

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Fig. 11.
Electrospray ionization tandem mass
spectrometric quantitation of islet lysophosphatidylcholine
content. Islets were treated with vehicle (A and
C) or 25 µM BEL (B) for 30 min at
37 °C and then incubated for 10 min at 37 °C without
(A and B) or with 50 units/ml bee venom
sPLA2 (C). Islets lipids were then extracted,
mixed with 300 pmol of internal standard 17:0-LPC, and analyzed by TLC
to isolate LPC, which was extracted from the plate and analyzed as
Li+ adducts by ESI/MS/MS with neutral loss of 59 m/z units scanning.
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Fig. 11 illustrates that the two most abundant LPC species in islets
are 16:0-LPC and 18:0-LPC, consistent with a previous report (63), that
treatment of islets with BEL induces a modest decline in the abundance
of these species relative to the internal standard, and that treatment
with bee venom sPLA2 induces the expected increase in
abundance of these species. A series of similar experiments indicated
that the resting islet LPC mass measured by this method (5.11 pmol/islet) corresponds closely to a measurement of 5.55 pmol/islet
based on GC/MS quantitation islet LPC fatty acid content (63) and that
the mean fall in LPC mass in BEL-treated islets is about 25% (Table
III, top). This corresponds closely to a
21% decrement induced by BEL in [3H]LPC levels in
[3H]choline-labeled islets (Table III, bottom). Both
methods indicate that BEL induces a smaller decline in islet LPC levels
than the 60% decrement for P388D1 cells (15, 16). Although BEL induces a modest decline in islet LPC content (Table III), this is not associated with reduced [3H]arachidonic acid
incorporation into islet PC (Fig. 7), suggesting LPC level does not
limit arachidonate incorporation into islet PC.
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Table III
Examination of islet lysophosphatidylcholine levels by electrospray
ionization tandem mass spectrometry and by radiochemical analyses with
[3H]choline labeling
Top, experiments were performed as in Fig. 11, and the quantity of
16:0-LPC (m/z 502) and 18:0-LPC (m/z 530) were
determined relative to the internal standard 17:0-LPC (m/z
516) by interpolation from a standard curve (n = 6).
Bottom, islets were incubated with [3H]choline for 40 h
at 37 °C and were treated with vehicle (Control) or with 25 µM BEL for 30 min at 37 °C. The islets were then
incubated for 10 min at 37 °C. At the end of the incubation, islet
lipids were extracted and analyzed by TLC to isolate LPC, which was
extracted from the plate. The 3H content of the LPC fraction
was determined by liquid scintillation spectrometry (n = 6).
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Although iPLA2 activity is not required for arachidonate
incorporation or phospholipid remodeling in islets, we have suggested that iPLA2 might participate in insulin
secretagogue-induced hydrolysis of arachidonic acid from islet
phospholipids, based on the finding that BEL suppresses insulin
secretagogue-induced release of arachidonate metabolites from islets
(81). To determine whether this might reflect inhibition of PAPH-1 (4,
57) rather than iPLA2, we compared effects of BEL and the
PAPH inhibitor propranolol (29, 70) on accumulation of nonesterified
arachidonate in islets stimulated with the insulin secretagogue 17 mM D-glucose alone or in combination with the
muscarinic agonist carbachol (Fig. 12).
As measured by isotope dilution GC/MS with
[2H8]arachidonate as internal standard, 17 mM D-glucose induced a doubling in accumulation
of nonesterified arachidonic acid in islet incubations (Fig. 12),
consistent with previous reports (82, 83), and this response was
greatly amplified by carbachol (Fig. 12). The effect of 17 mM glucose alone was completely prevented by BEL, and the
response to the combination of 17 mM glucose plus carbachol
was reduced by about 70%. Propranolol did not reduce accumulation of
nonesterified arachidonate induced either by 17 mM glucose
alone or by the combination of 17 mM glucose plus carbachol (Fig. 12).

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|
Fig. 12.
Effects of BEL and propranolol on insulin
secretagogue-induced accumulation of nonesterified arachidonic acid in
pancreatic islet incubations. Islets were treated with vehicle
(cross-hatched bars), 25 µM BEL
(stippled bars), or 250 µM propranolol
(dark bars) for 30 min at 37 °C. The islets were then
incubated (30 min at 37 °C) in medium containing 3 mM
glucose, 17 mM glucose, or 17 mM glucose plus
0.5 mM carbachol. Propranolol (250 µM) was
included in the incubation medium for islets that had been preincubated
with propranolol. At the end of the incubation, lipids were extracted,
mixed with 825 pmol of [2H8]arachidonic acid
internal standard, and analyzed by RP-HPLC to isolate arachidonic acid,
which was converted to a pentafluorobenzyl (PFBE) ester
derivative and analyzed by GC/MS in negative ion electron capture mode
with selected monitoring of the carboxylate anions of endogenous
arachidonate (m/z 303) and of the internal standard
(m/z 311). Endogenous arachidonate content was determined
from the ratio of the integrated areas of the two ion current peaks at
the appropriate retention time by interpolation from a standard curve
and normalized to islet protein content. Displayed values represent the
arachidonate content in each sample divided by the mean content in
islets that had been incubated with 3 mM glucose without
BEL or propranolol (n = 6). Conditions that differed
from the control with a p value of less than 0.05 are
denoted with an asterisk.
|
|
 |
DISCUSSION |
A housekeeping role for iPLA2 in phospholipid
remodeling has been suggested from iPLA2 inhibition studies
in P388D1 cells (4, 15, 16), but we find no evidence that
iPLA2 activity is required for incorporation of
arachidonate or phospholipid remodeling in pancreatic islets or
insulinoma cells. A difficulty in studying iPLA2 functions
is that the most effective pharmacologic inhibitor of the enzyme, BEL
(8-12), also inhibits PAPH-1 (57), although BEL does not inhibit
sPLA2 or cPLA2 enzymes at concentrations that
effectively inhibit iPLA2 (11-14, 57). One approach to
this problem is to compare effects of BEL and of the PAPH inhibitor propranolol to distinguish functional consequences of inhibition of
iPLA2 or of PAPH (29, 70), and a second employs antisense oligonucleotide suppression of iPLA2 expression (16). We
used the first approach here but were unable to suppress
iPLA2 activity in INS-1 insulinoma cells with antisense
oligonucleotides using conditions applied to P388D1 cells (16) and
several variations on them. It has been suggested that the efficacy of
antisense suppression may be affected by iPLA2 expression
level (29). Amniotic WISH cells, for example, apparently express much
higher iPLA2 levels than P388D1 cells (29), and our
inability to suppress INS-1 cell iPLA2 with antisense
oligonucleotides might reflect high iPLA2 expression.
Others have also encountered difficulties with lack of efficacy and
lack of specificity of antisense approaches (84), which may offer no
greater selectivity than conventional pharmacologic agents even when
suppression of the target is achieved (85).
Despite difficulties in achieving completely selective
iPLA2 inhibition, the fact that incorporation of
arachidonic acid into islet or INS-1 cell phospholipids is not
suppressed when iPLA2 is inhibited by 98% indicates that
iPLA2 activity is not required for this process in these
cells. Pathways for arachidonate incorporation into islet cells appear
to differ in several respects from those in P388D1 cells. Such
differences might be expected from the marked difference in the
esterified arachidonate content in islet phospholipids, which is among
the highest of any known tissue (37-39), compared with that of P388D1
macrophage-like cells, which contain far lower levels of esterified
arachidonate (27-29) than do native macrophages (25, 26) and higher
levels of nonesterified arachidonate (29) than other cells (30-32).
P388D1 cells may be deficient in arachidonate incorporation mechanisms
employed by other cells and forced to use mechanisms not employed by
cells that can maintain a high content of arachidonate-containing phospholipids.
Although arachidonate incorporation is unaffected by chelation of
extracellular or intracellular Ca2+ in P388D1 cells (15),
chelation of intracellular Ca2+ reduces but does not
eliminate incorporation of arachidonate and palmitate into islets, when
incorporation is compared in non-BAPTA-loaded islets and in
BAPTA-loaded islets incubated with 3H-fatty acid in
Ca2+-free EGTA-containing medium. This suggests that both
Ca2+-dependent and Ca2+-independent
processes participate in fatty acid incorporation into islet
phospholipids. Although Ca2+-independent hydrolysis of
phospholipids by iPLA2 to generate lysophospholipid
acceptors is proposed to be the Ca2+-independent event that
limits arachidonate incorporation into P388D1 cells (15, 16), only
about 60% suppression of arachidonate incorporation into P388D1 cells
is achieved under conditions where iPLA2 activity is
completely inhibited (15). This suggests that P388D1 cells also employ
Ca2+-independent mechanisms other than iPLA2 to
generate a substantial fraction of LPC acceptors that limit
arachidonate incorporation into these cells (15, 16).
Ca2+-independent mechanisms for generating
lysophospholipids that do not involve iPLA2 exist in other
cells (86). The Ca2+-dependent processes that
participate in fatty acid incorporation into islet cells have not yet
been identified, but islet beta cells express at least two classes of
Ca2+-dependent PLA2 enzymes that
might contribute to islet lysophospholipid content (14, 87).
The role of LPC content in regulating fatty acid incorporation into GPC
lipids and the contribution of iPLA2 to LPC levels also
differ between islets and P388D1 cells. BEL induces a more modest
reduction in LPC levels in islets than in P388D1 cells, and this
reduction does not impair arachidonate incorporation into islet PC but
does so in P388D1 cells (15, 16). This suggests that iPLA2
makes only a modest contribution to islet LPC content and that islet
LPC is maintained at sufficiently high levels by non-iPLA2-dependent mechanisms that
arachidonate incorporation into islet PC is not limited by LPC level.
ESI/MS/MS measurements here and previous measurements by other methods
(63) yield similar estimates of islet LPC content and indicate that LPC
levels in rat islets are substantially higher than that in rat liver
(53), and this may be one means employed by islets to maintain a higher content of arachidonate-containing phospholipids than liver (39) or
P388D1 cells (27-29). Although LPC levels do not limit arachidonate incorporation into islet phospholipids, our experiments with IL-1 here
and elsewhere (51) indicate that generation of arachidonoyl-CoA may
limit such incorporation under circumstances where production of ATP is
impaired or CoASH degradation is accelerated. IL-1 stimulates islet
production of nitric oxide, which impairs ATP generation by islet
mitochondria (68) and reacts with free thiol groups (69), such as those
of CoASH. Both ATP and CoASH are required to form arachidonoyl-CoA from
arachidonic acid (26, 33, 34), an obligatory step for its incorporation
into phospholipids (35, 36).
Although we never observe suppression of fatty acid incorporation into
islet or insulinoma cell phospholipids when iPLA2 activity is inhibited, stimulation of incorporation is often observed. The
simplest explanation is that net incorporation of fatty acid reflects
the difference between acylation and deacylation and that
iPLA2 inhibition reduces deacylation but does not affect acylation because iPLA2-catalyzed generation of
lysophospholipid acceptors is not required for fatty acid incorporation
into islet phospholipids. The similar effects of BEL on incorporation
of both arachidonate and palmitate are consistent with the fact
iPLA2 hydrolyzes phospholipids with palmitate substituents
at least as readily as those with arachidonate substituents (8, 88). Although palmitate incorporation into P388D1 cells has been taken to
reflect primarily de novo phospholipid synthesis (15),
palmitate incorporation into islet phospholipids under our conditions
proceeds predominantly by deacylation/reacylation because BEL impairs
de novo phospholipid synthesis but does not suppress
palmitate incorporation. Deacylation/reacylation reactions with
palmitate occur as rapidly as those with arachidonate in some cells
(76-80). We have not determined whether palmitate incorporated into
islet phospholipids under our conditions resides primarily in the
sn-1 or sn-2 position, but islets contain
substantial amounts of GPC species with palmitate as the
sn-2 substituent, including 16:0/16:0-GPC (39).
Deacylation/reacylation of phospholipid sn-1 substituents
also occurs (76-80), and iPLA2 can catalyze hydrolysis
of sn-1 substituents of phospholipids (8).
The fact that iPLA2 is not required for arachidonate
incorporation or phospholipid remodeling in beta cells suggests that it
may play other roles in these cells. Various signaling functions for
iPLA2 have been suggested (10, 17-21), and as demonstrated here and elsewhere (81-83), insulin secretagogues induce hydrolysis of
arachidonic acid from islet phospholipids. Our findings that insulin
secretagogue-induced accumulation of nonesterified arachidonate in
islet incubations is reduced by BEL corroborates the observation that
insulin secretagogue-induced release of arachidonate metabolites from
islets is reduced by BEL (81). The failure of the PAPH inhibitor
propranolol (29, 70) to block insulin secretagogue-induced accumulation
of nonesterified arachidonate in islet incubations suggests that PAPH
is not the BEL-sensitive target that participates in insulin
secretagogue-induced hydrolysis of arachidonate from islet
phospholipids, although others (4, 57) have suggested that this process
involves sequential actions of phospholipase D, PAPH, and diglyceride
lipase. Our findings are consistent with observations by others that
250 µM propranolol maximally inhibits islet PAPH but does
not block islet accumulation of nonesterified arachidonate and
stimulates insulin secretion (71). PA produced by phospholipase D and
hydrolyzed by PAPH may also be an unlikely source of arachidonic acid
in signaling events because PA from this route contains primarily
saturated or monounsaturated fatty acids and little arachidonate
(89).
Signaling roles have been proposed for iPLA2 in apoptosis
induced by the Fas/Fas ligand system in U937 cells (20), in apoptosis induced by Ca2+ store depletion in beta cells (90), and in
leukotriene production by granulocytes (17). The possibility that
iPLA2 plays different roles and is subject to different
mechanisms of regulation in different cells is suggested by the fact
that stimuli which induce iPLA2-catalyzed arachidonate
release and leukotriene production in human granulocytes fail to induce
these events in human lymphocyte lines, even though both classes of
cells express iPLA2 and leukotriene biosynthetic enzymes
(17, 21). The facts that the active form of iPLA2 is an
oligomer (8, 88), that multiple splice variants exist in some cells
(21, 41), and that heterooligomeric complexes among different splice
variants exhibit altered catalytic activity (21) suggest that
iPLA2 may be subject to complex regulatory mechanisms that
differ among cell types. Our findings that iPLA2 activity
is not required for arachidonate incorporation into beta cell
phospholipids, even though iPLA2 appears to participate in such incorporation in P388D1 cells (15, 16), is consistent with the
notion that the enzyme plays diverse roles in different cell types.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Mary Mueller for excellent
technical assistance, Dr. Simon Jones of Genetics Institute for
providing the iPLA2 antibody, and Dr. Christopher Newgard
of the University of Texas at Dallas-Southwestern for providing the
INS-1 cells.
 |
FOOTNOTES |
*
This research was supported by National Institutes of Health
Grant R37-DK-34388.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Box 8127, Washington
University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-8190; Fax: 314-362-8188; E-mail
jturk{at}imgate.wustl.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
PLA2, phospholipase A2;
BEL, bromoenol lactone suicide
substrate;
BSA, bovine serum albumin;
cPLA2, group IV
phospholipase A2;
ECL, enhanced chemiluminescence;
ESI, electrospray ionization;
GC, gas chromatography;
GPC, glycerophosphocholine;
HBSS, Hank's balanced salt solution;
iPLA2, group VI phospholipase A2;
LPC, lysophosphatidylcholine;
MS, mass spectrometry;
MS/MS, tandem mass
spectrometry;
NEM, N-ethylmaleimide;
NMMA, NG-monomethyl-L-arginine acetate;
NP-HPLC, normal phase-high performance liquid chromatography;
PA, phosphatidic acid;
PAPH, phosphatidate phosphohydrolase;
PAGE, polyacrylamide gel electrophoresis;
PC, phosphatidylcholine;
PE, phosphatidylethanolamine;
PI, phosphatidylinositol;
RP-HPLC, reverse
phase high performance liquid chromatography;
RT, reverse
transcriptase;
PCR, polymerase chain reaction;
IL, interleukin;
bp, base pair;
BAPTA-AM, 1,2-bis-(O-aminophenoxylethane-N,N,N',N'-tetraacetic
acid tetra(acetoxymethyl) ester.
 |
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