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(Received for publication, June 13,
1995; and in revised form, October 19, 1995) From the
Interleukin-1 (IL-1) impairs insulin secretion from pancreatic
islets and may contribute to the pathogenesis of insulin-dependent
diabetes mellitus. IL-1 increases islet expression of nitric oxide (NO)
synthase, and the resultant overproduction of NO participates in
inhibition of insulin secretion because NO synthase inhibitors, e.g.N Isolated pancreatic islets from rats and humans convert
endogenous arachidonic acid to oxygenated metabolites, and islet
eicosanoid synthesis is stimulated by insulin
secretagogues(1, 2, 3, 4, 5, 6, 7, 8
, 9, 10) .
The most abundant islet eicosanoids are the cyclooxygenase product
PGE Little is known about the
influence of IL-1 on islet 12-HETE production, but 12-lipoxygenase
products may promote insulin secretion from islet Several observations suggested the possibility that IL-1 might
suppress islet 12-HETE production and that this might contribute to
suppression of glucose-induced insulin secretion. Augmentation by IL-1
of islet NO production (14) results in inactivation of some
iron-sulfur enzymes (21, 22) including mitochondrial
aconitase(23) . The 12-lipoxygenases are iron-sulfur
enzymes(12) , and the platelet 12-lipoxygenase isoform has been
reported to be inhibited by NO(24) . We have therefore
characterized the effects of IL-1 on islet 12-HETE production in the
studies described here.
Under the conditions of these studies (up to
24 h incubation with 5 units/ml IL-1), IL-1 is not cytotoxic to islets,
and effects on insulin secretion are fully reversible upon removal of
IL-1 from medium and continued culture(29) . Lack of
cytotoxicity in our experiments is also reflected by normal islet
morphology, preservation of islet number, preservation of islet total
acid-ethanol-extractable insulin content, and constant
glyceraldehyde-3-phosphate dehydrogenase expression during incubation
with IL-1 and other additives for up to 24 h.
For immunoblotting analyses, islets (200
per condition) were cultured (0.4 ml of cCMRL, 24 h, 37 °C) with or
without IL-1 (5 units/ml) or IL-1 plus actinomycin D (1
µM) under 95% air, 5% CO
Figure 6:
Examination of islet RNA for content of
species encoding 12-lipoxygenase, nitric oxide synthase, and
glyceraldehyde-3-phosphate dehydrogenase as a function of time of
incubation with interleukin-1. Isolated islets were incubated for
various periods at 37 °C with IL-1 (5 units/ml), and total RNA was
isolated as described under ``Experimental Procedures.''
First strand cDNA was transcribed from the RNA template with avian
myeloblastosis virus reverse transcriptase. PCR was performed as
described under ``Experimental Procedures'' with primer sets
designed to amplify fragments of sequences encoding rat leukocyte-type
12-lipoxygenase (12-LO, upper panel), inducible nitric oxide
synthase (iNOS, middle panel), or glyceraldehyde-3-phosphate
dehydrogenase (GAPDH, lower panel). PCR products were analyzed
by 1% agarose gel electrophoresis and visualized with ethidium
bromide.
Figure 10:
Examination of islet RNA for content of
species encoding a cytosolic 85-kDa PLA2, a 14-kDa type II PLA2, nitric
oxide synthase, and glyceraldehyde-3-phosphate dehydrogenase after
incubation with interleukin-1 without or with NMMA. Isolated islets
were incubated (6 h, 37 °C) with no additions (lanes 1),
with IL-1 (5 units/ml) (lanes 2), or with IL-1 plus NMMA (0.5
mM) (lanes 3). At the end of incubations, total RNA
was isolated as described under ``Experimental Procedures.''
First strand cDNA was transcribed from the RNA template with avian
myeloblastosis virus reverse transcriptase. PCR was performed, as
described under ``Experimental Procedures,'' with primer sets
designed to amplify a fragment of sequences encoding
glyceraldehyde-3-phosphate dehydrogenase (GAPDH, first set of
3 lanes), inducible nitric oxide synthase (iNOS, second set of
3 lanes), 85-kDa cytosolic PLA
To determine whether treatment of pancreatic islets with IL-1
would suppress 12-HETE production, islets were incubated without or
with IL-1 (5 units/ml) for 24 h. Islets were then incubated in the
absence of IL-1 for 30 min with 3 mMD-glucose (basal
condition) or with 17 mMD-glucose plus 0.5 mM carbachol (stimulatory condition). Islet production of 12-HETE was
then measured by GC-NICI-MS and release of PGE
Figure 1:
Influence of incubation
with interleukin-1 on eicosanoid release and insulin secretion from
isolated pancreatic islets. Islets were incubated for 24 h without (open symbols) or with IL-1 (closed symbols) and then
resuspended in KRB medium supplemented with 3 mM glucose or 17
mM glucose plus 0.5 mM carbachol. Incubations were
then performed for 30 min at 37 °C. At the end of the incubation,
medium 12-HETE (panel A, triangles) was quantitated by
GC-NICI-MS; PGE
The time course of production of 12-HETE and
PGE
Figure 2:
Time course of eicosanoid and nitrite
release from islets incubated with interleukin-1. Islets were incubated
without (open symbols) or with IL-1 (closed symbols)
in cCMRL for various periods at 37 °C. At the end of the
incubation, medium content of 12-HETE (panel A, circles) was
quantitated by GC-NICI-MS; PGE
The effects of IL-1 to induce an increase in islet
PGE
Figure 3:
Influence of the nitric oxide synthase
inhibitor NMMA and actinomycin D (AcD) and cycloheximide on
interleukin-1-induced enhancement of islet production of eicosanoids
and nitrite. Islets were incubated without additions (control, first column), with IL-1 (5 units/ml) alone (second column),
with IL-1 plus NMMA (0.5 mM) (third column), with
IL-1 plus actinomycin D (1 µM) (fourth column),
or with IL-1 plus cycloheximide (CHX, 10 µM) (fifth column) in cCMRL for 24 h at 37 °C. At the end of
the incubation, medium content of 12-HETE (panel A), PGE
The possible involvement of NO in enhancing
islet 12-HETE production by IL-1 suggested that the 12-HETE might
derive from NO-dependent, nonenzymatic peroxidation of arachidonate
rather than from 12-lipoxygenase action. NO interacts with superoxide
anion to yield peroxynitrite, which, upon protonation, decomposes to
yield hydroxyl radicals which can initiate lipid
peroxidation(49) . The stereochemical composition of 12-HETE
generated from IL-1-treated islets was therefore determined. Islet
12-lipoxygenase produces exclusively 12-S-HETE, but nonenzymatic lipid
peroxidation produces a racemic mixture of 12-S- and
12-R-HETE(8) . Stereochemical analyses were performed
by addition of racemic [
Figure 4:
Chiral phase HPLC analysis of 12-HETE
released from islets incubated with interleukin-1. Islets were
incubated with IL-1 for 24 h in cCMRL at 37 °C. Medium 12-HETE was
then extracted, mixed with racemic
[
To evaluate the possibility that IL-1 increases expression of islet
12-lipoxygenase, conversion of exogenous arachidonate to 12-HETE by
islets incubated without or with IL-1 was examined. Previously,
incubation of islets with IL-1 was found to increase PGE
Figure 5:
Conversion of exogenous arachidonic acid
to eicosanoids by islets incubated with or without interleukin-1 and
immunoblotting analyses of islet 12-lipoxygenase protein. Islets were
incubated without (open symbols) or with IL-1 (closed
symbols) for 24 h at 37 °C in cCMRL. Islets were then
resuspended in nKRB containing either no exogenous arachidonic acid or
supplemented with 10, 50, or 100 µM arachidonic acid.
Incubations were performed for 20 min at 37 °C after addition of
arachidonic acid or vehicle. At the end of the incubation, medium
PGE
In addition,
RT-PCR experiments using primers specific for the sequence of rat
leukocyte-type 12-lipoxygenase and islet RNA as template yielded a
product of the expected size (632 base pairs) for a 12-lipoxygenase
cDNA fragment before addition of IL-1 (time 0 lane of upper panel of Fig. 6), but this material did not
increase in abundance after adding IL-1 to islets (time 2, 4, 8,
24, and 48 h lanes of upper panel of Fig. 6). Under these conditions, RT-PCR analyses using primers
specific for sequences of iNOS or for glyceraldehyde-3-phosphate
dehydrogenase revealed clear induction of iNOS mRNA and constant levels
of glyceraldehyde-3-phosphate dehydrogenase mRNA (middle and lower panels of Fig. 6). Activity and protein level
measurements and RT-PCR estimates of mRNA levels thus all indicate that
IL-1 does not induce islet 12-lipoxygenase synthesis. Potential
explanations for suppression of IL-induced enhancement of islet 12-HETE
production by NMMA are that NMMA inhibits or that NO activates islet
12-lipoxygenase. Effects of NMMA and the NO-releasing compound SIN-1
(3-morpholinosydnonimine) (21) on islet conversion of exogenous
arachidonate to 12-HETE were therefore examined. Preincubation of
islets with NMMA or with SIN-1 before addition of arachidonate did not
significantly affect islet conversion of exogenous arachidonate to
12-HETE (Fig. 7). This indicates that NMMA does not inhibit
islet 12-lipoxygenase and suggests that islet 12-lipoxygenase, in
contrast to the platelet isoform, is relatively insensitive to NO.
Figure 7:
Influence of the nitric oxide synthase
inhibitor NMMA or the nitric oxide-releasing compound SIN-1 on islet
conversion of exogenous arachidonic acid to 12-HETE. Islets were
incubated without (closed symbols) or with IL-1 (open
symbols) for 24 h at 37 °C in cCMRL. Islets were then
resuspended in nKRB containing NMMA at a concentration of 0.5 mM (open triangles), no NMMA (all other conditions), SIN-1
at a concentration of 100 µM (closed triangles),
or no SIN-1 (all other conditions) and were preincubated for 10 min at
37 °C. Medium was then supplemented with either no exogenous
arachidonic acid or with arachidonic acid at concentrations of 50 or
100 µM, and incubations were performed for 20 min at 37
°C. At the end of the incubation, medium content of 12-HETE was
quantitated by GC-NICI-MS. Values are expressed as nanograms of 12-HETE
per incubation. Error bars represent S.E. (n =
4).
The observations that IL-1 enhances islet production of 12-HETE by
12-lipoxygenase but does not increase expression of 12-lipoxygenase
enzyme suggest that IL-1 might increase substrate availability. Effects
of IL-1 on release of nonesterified arachidonate from islets were
therefore examined by isotope dilution GC-NICI-MS. Medium content of
arachidonic acid for control islets incubated with no additions for 24
h was found to be 306 ± 126 ng/ml and that for islets incubated
with IL-1 rose to 1109 ± 130 ng/ml (p < 0.001, Fig. 8, panel A). The NO synthase inhibitor NMMA
prevented IL-1-induced accumulation of nonesterified arachidonic acid (Fig. 8, panel A), under conditions where NMMA also
suppressed IL-1-induced accumulation of 12-HETE (Fig. 8, panel B) and PGE
Figure 8:
Release
of arachidonic acid and eicosanoids from islets incubated with or
without interleukin-1 in the presence or absence of the NO synthase
inhibitor NMMA. Islets were incubated (24 h, 37 °C, cCMRL) with no
additions (control, first column), with IL-1 alone (5
units/ml) (second column), or with IL-1 plus NMMA (0.5
mM) (third column). At the end of incubations, medium
arachidonic acid (panel A) and 12-HETE (panel B) were
quantitated by GC-NICI-MS and PGE
Consistent with a role for NO in accumulation of nonesterified
arachidonate is the observation that the NO-releasing compound SIN-1
induced accumulation of non-esterified arachidonate in medium of islets
incubated with this compound (Fig. 9). This effect was prevented
by the NO scavenger hemoglobin (21) but was not influenced by
the NO synthase inhibitor NMMA (Fig. 9). The lack of effect of
NMMA was expected because SIN-1 releases NO by a nonenzymatic process,
and NMMA is not a chemical scavenger of NO. The lack of effect of NMMA
on accumulation of nonesterified arachidonate in medium of islets
incubated with SIN-1 also suggests that NMMA does not inhibit islet
phospholipases which release arachidonic acid esterified in
phospholipids. NMMA also failed to suppress and SIN-1 failed to
activate hydrolysis of radiolabeled fatty acids from synthetic
phospholipid substrates catalyzed by Ca
Figure 9:
Influence of the nitric oxide-releasing
compound SIN-1 on islet arachidonate release. Islets were incubated in
cCMRL for 3 h at 37 °C after all additions were complete, and
medium content of arachidonate was then quantitated by GC-NICI-MS.
Control islets (first column) were incubated without
additions. SIN-1 was added at a final concentration of 100 µM to the remaining conditions (second through fourth
columns). For conditions involving agents (NMMA or hemoglobin (Hgb)) in addition to SIN-1, these agents were added 5 min
before addition of SIN-1. NMMA (third column) was added at a
final concentration of 500 µM. Hemoglobin (fourth
column) was added at a final concentration of 50 µM. Error bars represent S.E. (n =
3).
To examine the possibility that IL-1 might
increase expression of islet PLA In addition to cPLA To examine the possibility that ASCI-PLA A
mechanism whereby NO might increase availability of nonesterified
arachidonate without enhancing hydrolysis of arachidonate from
phospholipids is by suppressing re-esterification of arachidonate
released during phospholipid turnover. Evidence that incubation of
islets with IL-1 suppresses esterification of arachidonic acid into
phospholipids by a mechanism involving NO production was obtained from
experiments in which islets were pulse-labeled (15 min) with
[
Figure 11:
Influence of prior incubation with
interleukin-1 with or without NMMA on incorporation of
[
The
magnitude of the effect of prior IL-1-treatment to suppress
esterification of [
Figure 12:
Influence of prior incubation of
interleukin-1 with or without NMMA on the time course of incorporation
of [
Figure 13:
Normal
phase HPLC analysis of [
Figure 14:
Influence of oligomycin on incorporation
of [
Interleukin-1 impairs insulin secretion by pancreatic islets
and induces islet expression of cyclooxygenase and NO synthase,
resulting in overproduction of PGE Surprisingly, IL-1 enhanced rather than
suppressed islet 12-HETE production. This effect required several hours
to develop and was prevented by inhibitors of transcription and
translation, suggesting a requirement for new protein synthesis, and
was prevented by the NO synthase inhibitor NMMA. Stereochemical
analysis of 12-HETE from IL-1-treated islets indicated that it
consisted only of the S-enantiomer, reflecting enzymatic
synthesis and not nonenzymatic lipid peroxidation. Immunochemical and
RT-PCR studies indicated that IL-1 did not increase 12-lipoxygenase
protein or mRNA, and IL-1 failed to enhance islet conversion of
exogenous arachidonate to 12-HETE. NMMA did not impair islet conversion
of arachidonate to 12-HETE, indicating that NMMA does not inhibit islet
12-lipoxygenase. IL-1 did enhance release of nonesterified arachidonate
from islets and this effect was also suppressed by NMMA. These
observations suggest that enhanced 12-HETE production by IL-1-treated
islets reflects increased substrate availability to the 12-lipoxygenase
and that this effect occurs by an NO-dependent mechanism. This
possibility is supported by the fact that incubation of islets with an
NO-releasing compound enhanced accumulation of nonesterified
arachidonate and that this effect was prevented by an NO scavenger.
That this effect was not suppressed by NMMA suggests that NMMA does not
inhibit islet phospholipases, as does the failure of NMMA to inhibit
hydrolysis of fatty acids from phospholipid substrates catalyzed by
phospholipases in islet cytosol and membranes. IL-1 induces
synthesis of PLA Islets express an ATP-stimulated, Ca Both the time required
for IL-1 to enhance islet 12-HETE production and the requirement for
new protein synthesis may be attributable to induction of NO synthase
in islets by IL-1 (14, 23, 34, 58) because NO
production is required for enhanced 12-HETE production. Among the
mechanisms whereby NO might increase 12-HETE production is by
increasing 12-lipoxygenase substrate availability by suppressing
re-esterification of arachidonate released during phospholipid
turnover. Such re-esterification requires formation of arachidonyl-CoA
in a reaction that requires both ATP and CoASH. NO inhibits
mitochondrial iron-sulfur enzymes including aconitase, inhibits
mitochondrial respiration, and impairs ATP
generation(21, 23) . NO can also nitrosylate thiol
groups(84) . Prior incubation of islets with IL-1 impaired
islet incorporation of
[ A model consistent with our observations is
that there is an ongoing cycle of deacylation/reacylation of
arachidonate-containing phospholipids in islets. The deacylation
component of this cycle may be mediated by the HELSS-sensitive enzyme
ASCI-PLA The effect of IL-1 to
increase islet 12-HETE production may contribute to deleterious actions
of IL-1 on islets. 12-HETE is generated from the hydroperoxy precursor
12-HPETE; 12-HPETE induces apoptosis of cells with reduced levels of
glutathione peroxidase(86) ; and islets have low levels of
glutathione peroxidase(87) . Substrate-induced lipoxygenase
activity also induces superoxide generation in the presence of reduced
pyridine nucleotides, and this has been suggested to contribute to
cellular oxidative stress(88) . Islets also have low levels of
superoxide dismutase(89) , which reduces cellular superoxide
levels. Superoxide and NO react to yield peroxynitrite, which, when
protonated, yields hydroxyl radical(49) , a potent oxidant
postulated to participate in cytokine-induced islet
injury(15) . The effects of IL-1 to increase substrate flux
through the lipoxygenase and to augment NO production may therefore
interact cooperatively to inflict injury on beta cells. Note Added in Proof-We have also found
that rat pancreatic islets express mRNA for a 14-kDa type I PLA
Volume 271,
Number 2,
Issue of January 12, 1996 pp. 1029-1042
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-monomethyl-arginine (NMMA),
prevent this inhibition. While exploring effects of IL-1 on islet
arachidonic acid metabolism, we found that IL-1 increases islet
production of the 12-lipoxygenase product 12-hydroxyeicosatetraenoic
acid 12-(HETE). This effect requires NO production and is prevented by
NMMA. Exploration of the mechanism of this effect indicates that it
involves increased availabilty of the substrate arachidonic acid rather
than enhanced expression of 12-lipoxygenase. Evidence supporting this
conclusion includes the facts that IL-1 does not increase islet
12-lipoxygenase protein or mRNA levels and does not enhance islet
conversion of exogenous arachidonate to 12-HETE. Mass spectrometric
stereochemical analyses nonetheless indicate that 12-HETE produced by
IL-1-treated islets consists only of the S-enantiomer and thus
arises from enzyme action. IL-1 does enhance release of nonesterified
arachidonate from islets, as measured by isotope dilution mass
spectrometry, and this effect is suppressed by NMMA and mimicked by the
NO-releasing compound 3-morpholinosydnonimine. Although IL-1 increases
neither islet phospholipase A
(PLA) activities
nor mRNA levels for cytosolic or secretory PLA, a suicide
substrate which inhibits an islet Ca
-independent
PLA prevents enhancement of islet arachidonate release by
IL-1. IL-1 also impairs esterification of
[
H
]arachidonate into islet
phospholipids, and this effect is prevented by NMMA and mimicked by the
mitochondrial ATP-synthase inhibitor oligomycin. Experiments with
exogenous substrates indicate that NMMA does not inhibit and that the
NO-releasing compound does not activate islet 12-lipoxygenase or
PLA activities. These results indicate that a novel action
of NO is to increase levels of nonesterified arachidonic acid in
islets.
(
)and the 12-lipoxygenase prod
uct
12-hydroxy-(5,8,10,14)-eicosatetraenoic acid (12-HETE). PGE may negatively modulate insulin secretion (11) and exerts
proinflammatory effects(12) . Exposure of islets to
interleukin-1 (IL-1) suppresses glucose-induced insulin secretion and
augments islet PGE synthesis(13) , an effect
attributable to increased expression of the inducible isoform of
cyclooxygenase-2 and to activation of cyclooxygenase-2 by nitric oxide
(NO), which is overproduced by IL-1-treated islets(14) . The
latter effect reflects increased expression of the inducible isoform of
NO synthase by IL-1-treated islets(14) . Cytokines such as IL-1
are thought to participate in induction of islet dysfunction and
destruction in type I diabetes mellitus(15) , and both NO and
PGE are mediators which may adversely affect islet
function(16, 17) .
-cells. Fuel
insulin secretagogues stimulate islet 12-HETE production; exogenous
12-HPETE, the precursor of 12-HETE, stimulates insulin
secretion(1) ; and pharmacologic inhibitors of 12-lipoxygenase
suppress both 12-HETE production and insulin secretion with identical
concentration
dependences(1, 2, 3, 4, 5, 6, 7, 8) .
Islet 12-HETE consists exclusively of the S-enantiomer,
indicating enzymatic synthesis(9) , and immunochemical analyses
indicate that islet 12-lipoxygenase is selectively expressed in
-cells but not in 
-cells or in pancreatic exocrine
cells(18) . The -cell 12-lipoxygenase (18) corresponds to the leukocyte isoform(19) , which
has different catalytic properties, primary structure, and tissue
distribution compared to the platelet 12-lipoxygenase
isoform(20) . The leukocyte isoform is also expressed by
neuroendocrine cells in addition to islets(19) , further
suggesting a role for this 12-lipoxygenase isoform in secretory events.
Materials
Male Sprague-Dawley rats
(180-220 g) were from Sasco (O'Fallon, MO); collagenase
from Boehringer Mannheim (Indianapolis, IN); tissue culture medium
(CMRL-1066), penicillin, streptomycin, Hanks' balanced salt
solution, heat-inactivated fetal bovine serum, and L-glutamine
from Life Technologies, Inc. (Grand Island, NY). Pentex bovine serum
albumin (BSA, fatty acid free, fraction V) was from Miles Laboratories
(Elkhart, IN); rodent Chow 5001 from Ralston Purina (St. Louis, MO);
and D-glucose from the National Bureau of Standards
(Washington, D.C.). Arachidonic acid was obtained from NuChek Prep
(Elysian, MN), dissolved in ethanol just before use, and diluted in
albumin-free buffer(25, 26) . IL-1 was from
Cistron Biotechnology (Pine Brook, NJ); N-monomethyl-L-arginine acetate from
Calbiochem; actinomycin D from Sigma; and Tran
S-labeled
methionine (1117 Ci/mmol) from ICN (Costa Mesa, CA). PGE radioimmunoassay materials and
[H]12-HETE were from Cayman Chemical
(Ann Arbor, MI). Both [H]arachidonic
acid and [H]12-HETE (100 Ci/mmol)
were obtained from DuPont NEN.
[H]Arachidonic acid was prepared by
catalytic reduction of eicosa-(5,8,11,14)-tetrynoic acid (Hoffman
LaRoche) with [H]gas(27) .
Rabbit anti-12-lipoxygenase antiserum was a gift from Dr. Michael
Holtzman (Washington University, St. Louis), and its preparation and
properties have been described(18) .Media
Media included KRB (Krebs-Ringer bicarbonate
buffer; 25 mM HEPES, pH 7.4, 115 mM NaCl, 24 mM NaHCO, 5 mM KCl, 2.5 mM
CaCl, 1 mM MgCl); nKRB (KRB
supplemented with 3 mMD-glucose); cCMRL-1066
(CMRL-1066 from Life Technologies, Inc., supplemented with 10%
heat-inactivated fetal bovine serum, 1% L-glutamine, and 1%
(w/v) each of penicillin and streptomycin); and Hanks' balanced
salt solution from Life Technologies, Inc. (supplemented with 0.5%
penicillin-streptomycin).Isolation of Pancreatic Islets
Islets were
isolated aseptically from male Sprague-Dawley rats, as
described(28) , by collagenase digestion of excised, minced
pancreas, density gradient isolation, and manual selection under
microscopic visualization(28) . Isolated islets were
transferred into Falcon Petri dishes containing 2.5 ml of cCMRL-1066,
placed under an atmosphere of 95% air, 5% CO, and cultured
for various periods at 37 °C with or without IL-1 or other
additives.Incubation of Islets with IL-1 and Other
Additives
Islets (about 400/condition) were placed in Petri
dishes (10 
35 mm) and suspended in cCMRL medium (1 ml)
containing no additives, IL-1 (5 units/ml) alone, IL-1 plus NMMA
(0.5 mM), IL-1 plus actinomycin D (1 µM), or IL-1
plus CHX (10 µM). Incubations were then performed
(2-24 h, 37 °C). At the end of incubations, medium was
removed for measurement of PGE, 12-HETE, nitrite plus
nitrate, insulin, and arachidonic acid. In some experiments, islets
that had been incubated without or with IL-1 were removed from
incubation medium and their secretion of insulin and production of
eicosanoids examined in a subsequent incubation. In such experiments,
islets were washed (3 times, nKRB), transferred to siliconized test
tubes (10 75 mm), and preincubated (30 min, 37 °C) in nKRB
(0.2 ml). At the end of the preincubations, medium was removed and
islet incubated (30 min, 37 °C, under 5% CO, 95% air)
in KRB medium containing either 3 mMD-glucose or 17
mMD-glucose plus 0.5 mM carbachol. At the
end of that incubation, islet suspensions were placed in silanized
Sorvall centrifuge tubes (10 75 mm) and centrifuged (2000 rpm,
2 min). Eicosanoids were extracted separately from supernatant and
islet pellet (27) and measured. In other experiments, islets
that had been preincubated without or with IL-1 were incubated with
arachidonic acid (10-100 µM). In such experiments,
islets were removed from cCMRL, and 400 islets per condition were
resuspended in nKRB medium with no BSA. Arachidonic acid was then added
in ethanol (final ethanol concentration 0.1%) and incubations performed
(20 min, 37 °C). At the end of incubations, 12-HETE and PGE were extracted from medium and measured. Insulin content of islet
pellets was determined as described (3) after extraction with
75% ethanol, 1.5% HCl.Influence of the Nitric Oxide-releasing Compound SIN-1 on
Islet Arachidonate Release
Islets (about 400/condition) were
incubated in cCMRL (3 h, 37 °C) after additions were complete, and
medium content of arachidonate was then quantitated. Control islets
were incubated without further additions. SIN-1
(3-morpholinosydnonimine) was added (100 µM) to the
remaining conditions. For conditions involving agents (500 µM NMMA or 50 µM hemoglobin) in addition to SIN-1, these
agents were added 5 min before addition of SIN-1.Prostaglandin E
PGE
and Insulin
Measurement was extracted from acidified medium
with octadecylsilicic acid columns (1 ml, Baker Scientific,
Phillipsburg, NJ) and, after column washing, eluted with methyl
formate, as described(10, 30) . Eluant was
concentrated to dryness, reconstituted in buffer, and PGE
quantified by enzyme immunoassay, as described(30) . Insulin
content of islet incubation medium or acid-ethanol extracts of islet
pellets was measured by radioimmunoassay(31) .Nitrite Measurement
Medium nitrite content was
measured, after conversion of nitrate to nitrite with Aspergillus nitrate reductase(32, 33) ,
spectrophotometrically (540 nm, Titertek Multiskan MCC/340 microtiter
plate reader) after mixing medium (0.1 ml) with Griess reagent (0.1 ml
of a solution of 1 part of 1.32% sulfanilamide in 60% acetic acid and 1
part of 0.1% naphthylethylenediamine-HCl) and incubation (10 min, room
temperature) (23, 32, 34) .Quantitation of 12-HETE
12-HETE was quantitated by
stable isotope GC-MS in the negative ion chemical ionization (NICI)
mode (3, 4, 5, 6) . To aliquots of
medium, [H]12-HETE (20 ng) and
[H]12-HETE (50 nCi) were added as
internal standards, and medium was acidified (pH 3.0, 1 N HCl)
and extracted twice (CHCl). Extracts were
concentrated to dryness, and 12-HETE was converted to the
pentafluorobenzyl ester (PFBE) derivative. The 12-HETE-PFBE was
purified by reverse phase (RP)-HPLC on octadecylsilicic acid columns
(Ultrasphere ODS 4.6 250 mm, Alltech, Deerfield, IL) with the
solvent system (flow 1 ml/min) acetonitrile/water (70/30). The
12-HETE-PFBE peak (retention volume about 25 ml) was located by liquid
scintillation counting of aliquots of eluant, extracted
(CHCl), concentrated to dryness, converted to
the trimethylsilyl ether (TMS) derivative, and analyzed by GC-NICI-MS.
Quantitation of 12-HETE was performed relative to
[H]12-HETE internal standard by
reference to a standard
curve(3, 4, 5, 6) .Quantitation of Arachidonic Acid
Arachidonic acid
was quantitated by stable isotope dilution GC-NICI-MS(27) . To
aliquots of medium, [H]arachidonic
acid (100 ng), and [H]arachidonic
acid (50 nCi) were added as internal standards. Medium was acidified
(pH 3.0, 1 N HCl) and extracted twice
(CHCl). Extracts were concentrated to dryness,
reconstituted in HPLC mobile phase, and purified by RP-HPLC on an
Ultrasphere ODS column described above in the solvent system (flow 2
ml/min, column temperature 37 °C), acetonitrile/water/acetic acid
(80/20/0.1). The arachidonate peak was located by liquid scintillation
counting of column eluant (retention volume about 50 ml), extracted
(CHCl), concentrated to dryness, converted to a
PFBE derivative, and analyzed by GC-NICI-MS. Quantitation of
arachidonate was performed relative to
[H]arachidonate internal standard by
reference to a standard curve(27) .Derivatization
Carboxylate groups of 12-HETE or
arachidonic acid were converted to PFBE derivatives. To dry samples, 20
µl of N,N-dimethylacetamide/tetramethylammonium
hydroxide/methanol (10/5/15) and 20 µl of pentafluorobenzyl
bromide/N,N-dimethylacetamide () were added, and tubes were
vortex-mixed and incubated (room temperature, 15 min). After
concentration to dryness, water (50 µl) was added and vortex-mixed,
CHCl (200 µl) added and vortex-mixed, and
aqueous phase discarded. The PFBE derivative in the
CHCl phase was then concentrated to dryness and
reconstituted in mobile phase for HPLC analyses (12-HETE) or in heptane
for GC-MS analyses (arachidonate). Before GC-MS analyses, the hydroxyl
group of 12-HETE-PFBE was converted to a TMS ether derivative with N,O-bis(trimethylsilyl)trifluoroacetamide and pyridine (3, 4, 5, 6) .Preparation of
[
Racemic
[
O]12-HETE
O]12-HETE was prepared from
unlabeled, racemic 12-HETE (BIOMOL, Philadelphia, PA) by
butyrylcholinesterase (Type XI, Sigma)-catalyzed exchange in
HO and purified by RP-HPLC (8, 9) . The blank value ([
O]/[O]
content) of this material was 0.0035, as determined by GC-MS.Stereochemical Analysis of 12-HETE
Stereochemical
composition of 12-HETE was determined by sequential chiral phase HPLC
analysis and stable isotope dilution
GC-NICI-MS(8, 9) . 12-HETE from islet incubation
medium was extracted as above, mixed with racemic
[O]12-HETE (1 µg) internal
standard, concentrated to dryness, and converted to the PFBE
derivative, which was purified by RP-HPLC as above with flow-through UV
monitoring (235 nm) to locate the 12-HETE-PFBE peak. Appropriate
fractions were extracted (CHCl), concentrated
to dryness, reconstituted in mobile phase, and analyzed by chiral phase
HPLC with two Baker-Bond (dinitrobenzoylphenylglycine) ionically
coupled columns (4.3 25 mm) in series and a solvent system
(flow 0.8 ml/min) of hexane/isopropyl alcohol (1000/8). Approximate
retention times of 12-S-HETE-PFBE and 12-R-HETE-PFBE
were 30.8 and 31.8 min, respectively. Fractions (0.16 ml) were
collected in the region of interest, concentrated to dryness, converted
to TMS derivatives, and analyzed by GC-NICI-MS. The racemic internal
standard eluted as two peaks (S- and R-enantiomers)
of [O]12-HETE.Gas Chromatography
Derivatized samples of
12-HETE-(PFBE, TMS) or arachidonate-PFBE were introduced in heptane
into a Hewlett-Packard 5890 gas chromatograph (GC) via a Grob-type
injector (temperature, 225 °C) operated in the splitless mode and
analyzed on an HP Ultraperformance capillary column (8 m length,
cross-linked methylsilicone, inner diameter, 0.31 mm; film thickness,
0.17 µm) interfaced with a Hewlett-Packard 5988B mass
spectrometer(35, 36) . Helium was carrier gas (total
flow, 10 ml/min; head pressure, 4 lb/in). Initial oven
temperature was 85 °C. Injector and interface temperatures were 225
°C. At 0.5 min after injection, oven temperature was increased (40
°C/min) to a final temperature of 215 °C for analysis of
12-HETE-(PFBE, TMS) (retention time was about 5.78 min). For analysis
of arachidonate-PFBE, initial oven temperature was 85 °C, and 0.5
min after injection oven temperature was increased (30 °C/min) to a
final temperature of 200 °C (retention time about 5.49 min).Mass Spectrometry
The GC was interfaced with a
Hewlett-Packard 5988B mass spectrometer (MS) operated in NICI mode and
controlled by a Hewlett-Packard RTE-A data
system(35, 36) . Source temperature was 100 °C,
and methane was reagent gas (source pressure 1.5 torr). NICI mass
spectra of 12-HETE-(PFBE, TMS) and arachidonate-PFBE consist of a
single predominant ion corresponding to the carboxylate anion and
resulting from loss of the pentafluorobenzyl
moiety(3, 4, 27) . Analytes were quantitated
by selected monitoring of these ions, the m/z values of which
were 391 for endogenous 12-HETE-(PFBE, TMS), 395 for
[O]12-HETE-(PFBE, TMS), 399 for
[H]12-HETE-(PFBE, TMS), 303 for
endogenous arachidonate-PFBE, and 311 for
[H] arachidonate-PFBE.Immunochemical Analyses of Islet
12-Lipoxygenase
Immunoprecipitation was performed as described (14, 37) . Islets (100) were washed 3 times in
methionine-deficient MEM (MEM-Met: 9 parts MEM without methionine:1
part MEM with methionine) and incubated (5 h) without or with IL-1
(5 units/ml). [S]Methionine Trans-label (300
µCi) was added and islets incubated (13 h, 37 °C), isolated by
centrifugation, washed (3 times, 0.1 M PBS), and lysed (1 h, 4
°C) in 1 ml of TSA (0.01 M Tris-HCl, pH 8.0, 0.14 M NaCl, and 0.25% NaN) containing 1% Triton X-100, 1%
BSA, 1 mg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 1
mg/ml leupeptin, and 1 mM iodoacetamide. Cell debris was
removed (centrifugation, 30 min, 10,000 g, 4 °C)
and supernatants precleared (1 h) with cyanogen bromide-activated
protein A-Sepaharose (20 µl) (protein A-Sepharose swelled in TSA,
Sigma) diluted 1:1 with dilution buffer (0.1% Triton X-100 and 1% BSA
in TSA). Supernatants were cleared (centrifugation, 1 min, 4 °C,
200 g). Rabbit anti-12-lipoxygenase antiserum (1:500
dilution) and 40 µl of protein A-Sepharose were added and lysates
incubated (2 h, 4 °C). The anti-12-lipoxygenase antiserum is
directed against peptide CYLRPPSRVENSVAI from leukocyte-type
12-lipoxygenase and achieves immunoprecipitation of islet
12-lipoxygenase and recognizes it on immunoblotting(18) .
Protein A-Sepharose-antibody complexes were isolated by centrifugation
and washed twice with dilution buffer (1 ml), once with TSA (1 ml), and
once with buffer (1 ml, 0.05 M Tris-HCl, pH 6.8). Protein
A-Sepharose antibody complexes were treated with SDS sample mixture (30
µl, 0.25 M Tris-HCl, 20% -mercaptoethanol, 4% SDS)
and boiled (4 min) to dissociate complexes. Supernatants were obtained
by centrifugation and immunoprecipitates analyzed on 10%
SDS-polyacrylamide gels (38) and visualized by
fluorography(14) .. Islets were isolated
(centrifugation, 5000 g, 3 min) and washed 3 times
with PBS (0.5 ml, 0.1 M, pH 7.4). Samples were prepared for
electrophoresis by boiling (5 min) after adding HO (20
µl) and SDS sample mixture (30 µl) containing 5 mM EDTA and 1 mM EGTA. After SDS-polyacrylamide gel
electrophoresis, protein was transferred to nitrocellulose membranes.
12-Lipoxygenase was detected using the above antibody (dilution 1:1000)
and performing enhanced chemiluminescence according to the
manufacturer's instructions (Amersham).Reverse Transcriptase-Polymerase Chain Reaction Analyses
of Islet Content of mRNA Species
After islet incubation under
conditions described in Fig. 6and Fig. 10, total RNA was
isolated after solubilization in guanidinium thiocyanate by extraction
(phenol/chloroform/isoamyl alcohol) and precipitation (isopropyl
alcohol)(39) . First strand cDNA was transcribed with avian
myeloblastosis virus reverse transcriptase (Boehringer Mannheim). PCR
were performed on a Perkin-Elmer DNA Thermal Cycler 480. Amplification
steps (30 cycles) included denaturation (95 °C, 1 min), annealing
(50 to 60 °C, 1 min), and extension (72 °C, 2 min) performed in Taq polymerase buffer (Life Technologies, Inc.) containing 1.5
mM MgCl; 1 µM of each primer; 200
µM each of dATP, dGTP, dCTP, and dTTP; and 25 units/ml Taq DNA polymerase (Life Technologies, Inc.). PCR products
were analyzed by 1% agarose gel electrophoresis and visualized with
ethidium bromide(39) . Primer sets used to amplify fragments of
cDNA encoding specific gene products were: rat leukocyte-type
12-lipoxygenase(40) , sense 5`-AGGCACTCTGTTTGAAGC and antisense
5`-TTGAACATTCCCACCACG (expected fragment 632 bp); inducible nitric
oxide synthase(41) , sense 5`-TGCTTTGTGCGGAGTGTCAG and
antisense 5`-AGATGCTGTAACTCTTCTGG (expected fragment 650 bp);
glyceraldehyde-3-phosphate dehydrogenase(42) , sense
5`-TAGACAAGATGGTGAAGG and antisense 5`-TCCTTGGAGGCCATGTAG (expected
fragment 1006 bp); 85-kDa cytosolic PLA(43) , sense
5`-TGTTCAACAGAGTTTTGG and antisense 5`-AACAGAGCAACGAGATGG (expected
fragment 983 bp); 14-kDa type II PLA(44) , sense
5`-AGTTTGGGCAAATGATTCTG and antisense 5`-TCTTTCAGCAACTGGGCGTC (expected
fragment 372 bp).
(cPLA, third set
of 3 lanes), or a 14-kDa type II PLA (TIIPLA,
fourth set of 3 lanes). PCR products were analyzed by 1% agarose gel
electrophoresis and visualized with ethidium bromide. Similar results
were obtained for all of these PCR products in time course studies
similar to that in Fig. 6in which incubation time was varied
from 2 to 24 h. No induction of cPLA or TIIPLA was apparent at any time point, and
signal for
glyceraldehyde-3-phosphate dehydrogenase remained constant throughout
this period.
Subcellular Fractionation
Islet subcellular
fractionation was performed (45) after homogenization in buffer
(0.25 M sucrose, 10 mM histidine, pH 7.2) with a
Polytron apparatus (three bursts, 15 s, setting 6). Nuclei and cell
debris were removed by centrifugation (1000 g, 10
min). Mitochondrial pellets obtained after further centrifugation
(10,000 g, 10 min) were discarded and resultant
supernatants centrifuged (170,000 g, 60 min) to yield
membranous pellets and cytosolic supernatants. Membranous fractions
were resuspended in homogenization buffer.Phospholipase A
PLA Activity
Measurements activity in subcellular fractions
(150 µl of cytosolic or 100 µl of membranous fractions, average
protein content 70 µg) was assayed (45) by ethanolic
injection (5 µl) of 2.5 µM (final concentration)
radiolabeled phospholipid substrate in assay buffer (final conditions
400 µl of total volume, 200 mM Tris, pH 7.5, and either 10
mM EGTA or 10 mM CaCl). In experiments
examining effects of NMMA (0.5 mM) or SIN-1 (0.1 mM)
on PLA activity, test compounds were added 5 min before
addition of substrate. In experiments examining effects of prior
incubation with IL-1 on islet PLA activity, islets were
preincubated without or with IL-1 (5 units/ml, 24 h, 37 °C) before
preparation of subcellular fractions. Radiolabeled substrates were
1-0-(Z)-hexadec-1`-enyl-2-(9,10-[H])-octadec-9`-enoyl-sn-glycero-3-phosphoc
holine
or
1-hexadecanoyl-2-(1-[
C])-eicosa-5`,8`,11`,14`-tetraenoyl-sn-glycero-3-phosphoethanolamine.
Assay mixtures were incubated (2 min, 37 °C) and reactions
terminated by adding butanol (100 µl) (46) and vortexing.
The organic phase was separated by centrifugation (2000 g, 2 min) and a 25-µl aliquot applied to channeled Silica
Gel G TLC plates, which were then developed with petroleum ether/ethyl
ether/acetic acid (70/30/1) to resolve fatty acids (R
0.58) from diglycerides (R 0.
21-0.24).
The fatty acid region was scraped into a scintillation vial, and liquid
scintillation spectrometry was performed after addition of Universol (3
ml). PLA specific activity was calculated as
[R/(PxT)], where R is fatty acid released in picomoles, P
assay tube protein content (mg), and T assay duration (2 min).
Parameter R is [4 D S], where the factor 4
accounts for the fraction (25%) of butanol extracts analyzed; S is
specific radioactivity (dpm/pmol) of phospholipid substrate; and D is
dpm of released fatty acid. Parameter D is [(S - B)/E],
where S is assay sample cpm, B the blank cpm value for fatty acid
released with no added enzyme, and E the counting efficiency for
[H] (about 0.4) or [C]
(about 0.9).Protein Measurement
Protein content was measured
with Coomassie reagent against BSA as standard according to
manufacturer's instructions (Pierce).Influence of Prior Incubation with Interleukin-1 or
Oligomycin on Incorporation of
[
Islets were incubated without or with IL-1 (5 units/ml)
plus NMMA (500 µM), or with IL-1 (5 units/ml) alone (24 h,
37 °C) in cCMRL. In other experiments, islets were first incubated
without or with oligomycin (10 µg/ml) (1 h, 37 °C). Islets were
then removed from medium and resuspended (cCMRL) in Petri dishes (10
H
]Arachidonic Acid into Islet
Lipids 35 mm) to which were added 10 µCi (for IL-1 experiments) or
0.5 µCi (for oligomycin experiments) of
[H]arachidonic acid and unlabeled
arachidonic acid (10 µM). Dishes were then incubated (15
or 90 min, 37 °C) and islets transferred to 5-ml conical test tubes
and washed 3 times by suspension in and centrifugation from PBS plus
0.1% BSA to remove unincorporated radiolabel. Islet lipids were then
Bligh-Dyer extracted(47) . Extracts were concentrated to
dryness in scintillation vials, scintillant added, and H-content determined by liquid scintillation spectrometry.Normal Phase HPLC Separation of Islet Phospholipids into
Head Group Classes
Phospholipid extracts were concentrated to
dryness, reconstituted in hexane/isopropyl alcohol (1/1) (0.2 ml), and
analyzed by NP-HPLC (35) on a silicic acid column (LiChrospher
Si-100, 10 µ, 250 4.6 mm, Alltech) with a solvent system
gradient (flow rate 2 ml/min) of 100% A to (40% A, 60% B) over 30 min
followed by isocratic elution with flow-through UV monitoring (205 nm).
Solvent system A is hexane/isopropyl alcohol/water (485/485/30) and
solvent system B is hexane/isopropyl alcohol/water (465/465/70). H-Contents of aliquots of fractions were determined by
liquid scintillation spectrometry. Standard phospholipids exhibited the
retention times: phosphatidylethanolamine, 6 min; phosphatidic acid, 15
min; phosphatidylinositol, 19 min; phosphatidylserine, 28 min; and
phosphatidylcholine, 57 min.Influence of Incubation with Interleukin-1 on Retention
of Previously Incorporated
[
Islets were first prelabeled with
[H]Arachidonic
Acid in Islet LipidsH]arachidonic acid (10 µCi/ml)
in cCMRL (30 min, 37 °C) and washed 3 times (cCRML) to remove
unincorporated radiolabel. Islets were then resuspended in fresh
cCRMRL, counted (700/condition) into Petri dishes (10 35 mm),
and incubated (24 h, 37 °C) with no additions (control), with IL-1
(5 units/ml) alone, or with IL-1 (5 units/ml) plus NMMA (500
µM). At the end of incubations, islets were washed in PBS
plus 0.1% BSA to remove unincorporated radiolabel, and islet lipids
were Bligh-Dyer extracted (47) and their H-content
determined by liquid scintillation spectrometry.Reverse Phase HPLC Analysis of
[
In experiments described in Table 1,
[H]Arachidonate Released
from Prelabeled Islets Treated with Interleukin-1 and Other
AgentsH]arachidonate released from islets that had
previously been labeled and then incubated (24 h) without or with IL-1
in the presence or absence of NMMA at 5 or 20 mM glucose was
determined by RP-HPLC. Medium was acidified (pH 3.0, 1 N HCl),
extracted (CHCl), concentrated to dryness,
reconstituted in CHOH (0.1 ml), and analyzed on the
Ultrasphere ODS column described above in the solvent system (flow 2
ml/min, column temperature 40 °C) methanol/water/acetic acid
(80/20/0.1). The H-content of the arachidonate peak
(retention volume about 50 ml) was determined by liquid scintillation
spectrometry.
and insulin
by immunoassay. As illustrated in Fig. 1, 17 mMD-glucose plus carbachol stimulated insulin secretion and
production of PGE and 12-HETE by control islets, and prior
IL-1 exposure augmented PGE production and suppressed
insulin secretion, as previously
observed(3, 4, 5, 6, 8, 9, 13, 14<
/a>, 16) .
Surprisingly, prior IL-1 exposure did not suppress but rather enhanced
islet 12-HETE production (Fig. 1, panel A). The
majority of 12-HETE and PGE produced under these conditions
was released into medium, and little remained associated with islets
(not shown).
(panel B, squares) by enzyme
immunoassay; and insulin (panel C, circles) by
radioimmunoassay, as described under ``Experimental
Procedures.'' Displayed values for each parameter represent the
fold-increase over the mean control value (at 3 mM glucose for
vehicle-treated islets) and are means of duplicate determinations.
Absolute mean values for contents of 12-HETE and PGE in
supernatants from the control condition (3 mM glucose for
vehicle-treated islets) were 1.98 and 7.98 pmol, respectively, and the
mean total acid-extractable insulin content of control condition islet
pellets was 37.5 nmol.
was examined with islets incubated without or with IL-1
for various periods (Fig. 2). Incubation of islets with IL-1
induced a time-dependent rise in medium 12-HETE, PGE, and
nitrite contents compared to control conditions. Nitrite is an NO
oxidation product formed in aqueous solutions(21) , and its
accumulation reflects induction of islet nitric oxide synthase by
IL-1(14) . No difference in medium 12-HETE content, relative to
control conditions, was observed until islets had been incubated with
IL-1 for 8 h. IL-1-induced increases in medium PGE,
12-HETE, and nitrite contents continued to rise for 24 h, by which time
IL-1-treated islets had released nearly 10-fold more 12-HETE than
control islets.
(panel B, squares)
by enzyme immunoassay; and nitrite (panel C) by
spectrophotometric measurement after conversion of nitrate to nitrite,
as described under ``Experimental Procedures.'' Displayed
values for each parameter represent the fold-increase over the mean
control value (for vehicle-treated islets at each individual time
point). Error bars represent S.E. (n = 3).
Absolute mean values for contents of 12-HETE, PGE, and
nitrite in supernatants for the 2-h control condition were 3.13 pmol,
9.86 pmol, and 3.88 nmol, respectively, and mean total acid-extractable
insulin content of 2 h control condition islet pellets was 7.87
nmol.
production and to suppress insulin secretion require
new protein synthesis and are prevented by inhibitors of transcription (e.g. actinomycin D) or translation (e.g. CHX)(14, 48) . Islet proteins induced by IL-1
include inducible NO synthase and cyclooxygenase-2.(14) .
IL-1-induced enhancement of islet PGE production is also
partially suppressed by the NO synthase inhibitor NMMA, an effect
attributable to activation of cyclooxygenase by NO(14) . We
reasoned that IL-1-enhancement of islet 12-HETE production ( Fig. 1and Fig. 2) might reflect induction of synthesis of
12-lipoxygenase enzyme, analogous to its effects on cyclooxygenase-2,
but that the increase in 12-HETE synthesis might be blunted by
concomitant generation of NO, which was expected to partially inhibit
islet 12-lipoxygenase by analogy with platelet
12-lipoxygenase(24) . Effects of NMMA, actinomycin D, and CHX
on IL-1-stimulation of islet production of eicosanoids and nitrite were
therefore examined (Fig. 3). As expected, IL-1 enhancement of
islet production of PGE and nitrite was prevented by
actinomycin D and CHX and was reduced by NMMA. Both actinomycin D and
CHX also prevented IL-1-enhancement of islet 12-HETE production,
consistent with a requirement for protein synthesis for this effect.
Contrary to expectations, NMMA also prevented IL-1-stimulation of
12-HETE production (Fig. 3), suggesting that this effect
required NO generation.
(panel B), and nitrite (panel C) were
quantitated as described in the legend to Fig. 2. Displayed
values represent the fold-increase over the mean control value
(vehicle-treated islets). Error bars represent S.E. (n = 14). Mean values for contents of 12-HETE,
PGE, and nitrite in supernatants for the control condition
were 16.3 pmol, 90.6 pmol, and 20.3 nmol, respectively, and mean total
acid-extractable insulin content of control condition islet pellets was
8.36 nmol.
O]12-HETE
internal standard and sequential chiral-phase HPLC and then GC-NICI-MS
analyses. These analyses revealed that 12-HETE released from
IL-1-treated islets consisted exclusively of the S-enantiomer (Fig. 4) and established that the dominant mechanism in
enhancing islet 12-HETE production by IL-1 is enzymatic synthesis.
O]12-HETE internal standard,
converted to the PFBE derivative, purified by reverse phase HPLC, and
analyzed by chiral phase HPLC.
[O]12-HETE rather than
[H]12-HETE must be used as internal
standard because the latter but not the former separates from unlabeled
12-HETE on chiral phase HPLC analysis(8, 9) .
12-HETE-PFBE in individual fractions was collected separately,
converted to the TMS derivative, and analyzed by GC-NICI-MS. Racemic
[O]12-HETE-(PFBE, TMS) was
visualized by monitoring the ion at m/z 395 (open
symbols). The internal standard eluted as two peaks corresponding
to S-enantiomer (earlier peak) and R-enantiomer
(later peak). The endogenous, islet-derived
[O]12-HETE derivative was visualized
by monitoring the ion at m/z 391 (closed symbols) and
eluted as a single peak corresponding to the S-enantiomer.
production from maximally effective arachidonate
concentrations(13) , and this was the first evidence that IL-1
induces islet cyclooxygenase expression, a possibility later verified
by cyclooxygenase-2 immunochemical analyses(14) . Incubation of
islets with IL-1 for 24 h resulted in an enhanced ability to convert
exogenous arachidonate to PGE (Fig. 5, panel
A). In contrast, although exogenous arachidonate induced a
striking rise in islet 12-HETE production, no enhancement of this
effect was observed in IL-1-treated islets (Fig. 5, panel
B), suggesting that IL-1 did not increase expression of islet
12-lipoxygenase enzyme. This was supported by immunochemical analyses.
In immunoprecipitation studies, islets were metabolically labeled with
[S]methionine in the absence or presence of
IL-1, homogenized, and treated with anti-12-lipoxygenase antibody. Upon
analysis of immunoprecipitates by SDS-polyacrylamide gel
electrophoresis and fluorography, a S-labeled protein of
appropriate size (74 kDa) for 12-lipoxygenase was visualized, but IL-1
did not influence levels of this protein (not shown). Similar results
were obtained in immunoblotting studies of islet 12-lipoxygenase (inset in panel B, Fig. 5).
(panel A) and 12-HETE (panel B) were
quantitated as described in the legend to Fig. 2. Values for
PGE are expressed as picograms per incubation and values
for 12-HETE are expressed as nanograms per incubation. Error bars represent S.E. (n = 6). In the inset in panel B, immunoblotting analyses of 12-lipoxygenase protein
were performed as under ``Experimental Procedures'' for
islets that had been incubated for 24 h without additives (control, left lane), with IL-1 (5 units/ml) alone (middle lane), or with IL-1 plus actinomycin D (AcD,
1 µM, right lane). No difference in expression of
12-lipoxygenase protein was observed among these conditions. Analyses
displayed in the inset of panel B are representative
of three similar experiments.
(Fig. 8, panel
C). This suggests that enhanced 12-HETE production by IL-1-treated
islets reflects increased substrate availability to the 12-lipoxygenase
and that this effect is mediated by an NO-dependent mechanism.
(panel C) by
immunoassay. Values for arachidonate represent absolute mass (ng).
Values for 12-HETE and PGE represent the fold-increase over
control values. Error bars represent S.E. (n =
9). Mean values for control condition medium content of arachidonate,
12-HETE, and PGE were 1009, 34.6, and 133.2 pmol,
respectively, and mean acid-extractable insulin content of islet
pellets was 14.3 nmol.
-dependent or
Ca-independent PLA activities in islet
cytosolic or membranous fractions (not shown). This suggests that islet
PLA enzymes are neither directly inhibited by NMMA nor
directly activated by NO.
enzymes, both
Ca-dependent and Ca-independent
PLA activities were measured with exogenous phospholipid
substrates added to cytosolic or membranous fractions from control
islets and from islets that had been incubated with IL-1 for 24 h. IL-1
did not induce increases in any PLA activity in either
subcellular fraction (not shown). In addition, RT-PCR studies were
performed with islet RNA as template and primers specific for cytosolic
85-kDa PLA (cPLA), for a type II 14-kDa
PLA (TIIPLA), for iNOS, and for
glyceraldehyde-3-phosphate dehydrogenase. Under conditions where IL-1
induced an increase in iNOS mRNA, there was a constant level of signal
for message for cPLA, TIIPLA, and
glyceraldehyde-3-phosphate dehydrogenase (Fig. 10). Activity
measurements and RT-PCR analyses thus suggest that IL-1 does not
increase islet expression of cPLA or TIIPLA. and TIIPLA, a third
PLA activity expressed in islets is an ATP-stimulated,
Ca-independent
(ASCI)-PLA(31, 35, 36, 45, 50
) .
The molecular mass of the catalytic subunit of the islet enzyme (49) and of an analogous myocardial enzyme (51) is 40
kDa, and myocardial ASCI-PLA is activated by a regulatory
subunit which is an isoform of the glycolytic enzyme
phosphofructokinase(52) . Both islet and myocardial
ASCI-PLA are inhibited by a haloenol lactone suicide
substrate (HELSS) which does not inhibit cPLA or
TIIPLA(53, 54) . NO has recently been
reported to activate an ASCI-PLA in RAW 264.7 cells because
NO augments release of [H]arachidonic acid from
prelabeled cells; this effect is enhanced by increasing medium glucose
concentration; and the effect is suppressed by HELSS(55) .
ASCI-PLA activation in this system is thought to reflect
stimulation of glycolytic flux by NO(55) , perhaps because of
impairment of mitochondrial function by NO(21, 23) . act
ivation
might be responsible for IL-1-induced enhancement of islet release of
nonesterified arachidonate, islets were prelabeled by a 24-h incubation
with [H]arachidonate. Prelabeled
islets were then incubated with no additions, with IL-1 alone, or with
IL-1 plus NMMA in the presence of either 5 or 20 mM glucose
for 24 h. At the end of the incubation, release of
[H]arachidonic acid was measured by RP-HPLC. At 5
mM glucose, IL-1 induced more than a doubling of
[H]arachidonic acid release, and this effect was
prevented by NMMA (Table 1, panel A). IL-1-induced release of
[H]arachidonic acid was not enhanced by 20 mM glucose, however, but was attenuated (Table 1, panel A),
suggesting that enhanced glycolytic flux may not contribute strongly to
IL-1-induced enhancement of [H]arachidonic acid
release. Pretreatment of islets with the ASCI-PLA suicide
substrate HELSS prevented IL-1-induced enhancement of
[H]arachidonic acid release (Table 1, panel
B), suggesting that a HELSS-sensitive enzyme plays at least a
permissive role in IL-1-induced arachidonate release. Under these
conditions, HELSS pretreatment resulted in virtually complete immediate
suppression of islet cytosolic and membranous
Ca-independent PLA activities, although
activity recovered to 15-25% of control values after 24 h (Table 1, panel C). Incubation of islets with IL-1 for 24 h did
not induce an increase in Ca-independent PLA activities in islet cytosol or membranes (Table&nb
sp;1, panel
C), suggesting that IL-1 does not increase islet levels of
Ca-independent PLA enzymes.H]arachidonic acid after incubation
for 24 h with no additions, with IL-1 alone, or with IL-1 plus the NO
synthase inhibitor NMMA (Fig. 11, panel A).
[H]Arachidonate was incorporated
equally well into lipids of islets that had been incubated with no
additions or with IL-1 plus NMMA, but incorporation was substantially
reduced with islets incubated with IL-1 alone. (Under loading
conditions in Fig. 11, panel A, a mean specific
radioactivity of 517 dpm/fmol was achieved in the islet nonesterified
arachidonate pool, and there was no significant difference in this
parameter among the 3 conditions). Conversely, when islets were first
pulse-labeled with [H]arachidonate
and then incubated for 24 h with no additions, with IL-1 alone, or with
IL-1 plus NMMA, retention of [H]arachidonate in
islet lipids was indistinguishable for islets incubated with no
additions or with IL-1 plus NMMA but was significantly reduced for
islets incubated with IL-1 alone (Fig. 11, panel B).
The decline in content of esterified
[H]arachidonate in IL-1-treated
islets was also associated with a decrement in esterified arachidonate
mass (control 34.90 ± 4.7 pmol/islet; IL-1 24.65 ± 0.25
pmol/islet; IL-1 + NMMA 39.38 ± 3.97 pmol/islet).
H]arachidonic acid into islet lipids and on
retention of previously incorporated
[H]arachidonic acid in islet lipids. In panel
A, islets were incubated (24 h, 37 °C, cCMRL) with no
additions (control, left column), with IL-1 (5 units/ml) alone (IL-1, middle column), or with IL-1 (5 units/ml) plus NMMA
(500 µM) (IL-1 + NMMA, right column). Islets
were then removed from media, resuspended in fresh cCMRL, and counted
into Petri dishes. To each dish was then added
[H]arachidonate (10 µCi) mixed
with unlabeled arachidonate (final concentration 10 µM).
Dishes were then incubated (15 min, 37 °C). Islets were then washed
with PBS + 0.1% BSA to remove unincorporated radiolabel, and islet
lipids were extracted and their H-content determined. Error bars indicate S.E. (n = 6). The double asterisk over the middle column indicates
a
significant difference (p = 0.006) between the H-lipid content of islets that had been incubated with IL-1
compared to that of control islets. There was no significant difference (p = 0.269) between the H-lipid content of
control islets compared to that of islets that had been incubated with
IL-1 + NMMA. In panel B, islets were first prelabeled
with [H]arachidionic acid in cCMRL
(10 µCi/ml) for 30 min at 37 °C and then washed with cCRML to
remove unincorporated radiolabel. Islets (700/condition) were then
resuspended in fresh cCRMRL and incubated (24 h, 37 °C) with no
additions (control, left column), with IL-1 (5 units/ml) alone (middle column), or with IL-1 (5 units/ml) plus NMMA (500
µM, right column). At the end of incubations,
islets were washed in PBS plus 0.1% BSA to remove unincorporated
radiolabel, and islet lipids were extracted and their H-content determined. Error bars represent S.E. (n = 3). The double asterisk over the second
column indicates a significant difference (p = 0.001) between the H-lipid content of islets
that had been incubated with IL-1 compared to that of control islets.
There was no significant difference (p = 0.89) between
the H-lipid content of control islets compared to that of
islets that had been incubated with IL-1 +
NMMA.
H]arachidonate into islet
lipids became more prominent as the labeling period was extended from
15 to 90 min, and this effect was prevented at both time points if
exposure to IL-1 had occurred in the presence of NMMA (Fig. 12).
After the 90-min labeling period, islet phospholipids were extracted
and analyzed by NP-HPLC to separate phospholipid head group classes.
Under these short-term labeling conditions, the majority of radiolablel
is incorporated into phosphatidylcholine (Fig. 13), as
previously reported (56) . Prior treatment with IL-1 reduced
incorporation of [H]arachidonic acid into all
phospholipids except phosphatidylethanolamine, and the largest
decrement occurred in phosphatidylcholine (Fig. 13). (Total
phospholipid amounts recovered from control and IL-1-treated islets
under these conditions, as estimated by NP-HPLC-UV absorbance tracings,
were not different (not shown).) The fact that the effect of IL-1 to
suppress esterification of [H]arachidonic acid
into islet phospholipids was prevented by NMMA indicated that NO
production is required for this effect. Because arachidonate requires
ATP-dependent conversion to a coenzyme A thioester before
esterification into phospholipids and because NO inhibits mitochondrial
ATP generation(21, 23) , it is possible that
suppression of arachidonate esterification in IL-1-treated islets is
attributable to induction of islet NO synthase by IL-1 with resultant
NO overproduction and inhibition of mitochondrial function. Consistent
with this possibility, treatment of islets with the mitochondrial
ATP-synthase inhibitor oligomycin (57) was also found to impair
esterification of [H]arachidonic acid into islet
lipids (Fig. 14).
H] arachidonic acid into islet lipids.
Isolated islets were incubated (24 h, 37 °C, cCMRL) with no
additions (open circles), with IL-1 (5 units/ml) alone (closed triangles), or with IL-1 (5 units/ml) plus NMMA (500
µM) (closed circles). Islets were then removed
from media, resuspended in fresh cCMRL, and counted into Petri dishes.
To each dish was then added
[H]arachidonate (10 µCi) mixed
with unlabeled arachidonate (final concentration 10 µM).
Dishes were then incubated (15 or 90 min, 37 °C). Islets were then
washed with PBS + 0.1% BSA to remove unincorporated radiolabel,
and islet lipids were extracted and their H-content
determined. Error bars indicate S.E. (n =
3).
H]arachidonate
incorporation into phospholipid head group classes from islets that had
been previously incubated without or with interleukin-1. Isolated
islets were incubated (24 h, 37 °C, cCMRL) with no additions (solid line) or with IL-1 (5 units/ml) (dashed line).
Islets were then removed from media, resuspended in fresh cCMRL, and
counted into Petri dishes. To each dish was added
[H]arachidonate (10 µCi) and
unlabeled arachidonate (final concentration 10 µM). Dishes
were then incubated (90 min, 37 °C). Islets were then washed with
PBS + 0.1% BSA to remove unincorporated radiolabel, and islet
lipids were extracted and analyzed by NP-HPLC, as described under
``Experimental Procedures.'' The H-content of
eluant fractions was determined by liquid scintillation counting.
Elution positions of standard phospholipids are indicated (PE,
phosphatidylethanolamine; PA, phosphatidic acid; PI,
phosphatidylinositol; PS, phosphatidylserine; PC,
phosphatidylcholine).
H] arachidonic acid into islet lipids.
Isolated islets were incubated (cCMRL, 1 h, 37 °C) with no
additions (left column) or with oligomycin (10 µg/ml) (right column). Islets were then removed from media,
resuspended in fresh cCMRL, and counted into Petri dishes. To each dish
was then added [H]arachidonate (0.5
µCi) and unlabeled arachidonate (final concentration 10
µM). Dishes were then incubated (15 min, 37 °C).
Islets were then washed with PBS + 0.1% BSA to remove
unincorporated radiolabel, and islet lipids were extracted and their H-content determined. Error bars indicate S.E. (n = 3). The asterisk over the se
cond column
indicates that the p value for the difference from the control
condition is 0.028 by Student's t test.
and
NO(13, 14) . Inhibition of insulin secretion by IL-1
is attributable in part to islet NO production because this effect is
prevented by NO synthase inhibitors, e.g. NMMA(58) .
NO is thought to inhibit insulin secretion by inactivating iron-sulfur
enzymes including mitochondrial aconitase(23, 34) .
Because lipoxygenases are iron-sulfur enzymes that may complex with NO (59) and because islet 12-lipoxygenase products may promote
insulin
secretion(1, 2, 3, 4, 5, 6, 7, 8
, 9, 18) ,
we have characterized effects of IL-1 on islet production of the
12-lipoxygenase product 12-HETE to determine whether inhibition of
islet 12-lipoxygenase might be among the mechanisms whereby IL-1
inhibits insulin secretion. enzymes in some
cells(60, 61, 62) , and overexpression of
such enzymes in islets could result in increased levels of
nonesterified arachidonate. An 85-kDa cytosolic,
Ca-activated PLA (cPLA)
participates in arachidonate release in some cells (63, 64, 65, 66, 67) and is
expressed in human islets(68) . Immunochemical evidence
indicates that cPLA is expressed at very low levels in rat
islets(69) . Our RT-PCR data indicate that cPLA
mRNA is expressed in rat islets but is not induced by IL-1. Low
molecular mass (14 kDa) Ca-dependent PLA enzymes also exist in islets(70) . Our R
T-PCR data
indicate that islets express mRNA for a 14-kDa type II PLA,
but IL-1 treatment does not increase its abundance. Activity
measurements also failed to demonstrate increased expression of
Ca-dependent PLA activities in
subcellular fractions from IL-1-treated islets. Several enzymes may
contribute to activity in such
assays(71, 72, 73, 74, 75, 76, 77, 78, 79, 80) ,
however, which could obscure induction of a specific enzyme. Activation
of PLA by reversible phosphorylation (67, 81) or by NO-amplified Ca
entry (82) might also not be demonstrable with broken cell assays. -independent
PLA (ASCI-PLA) which participates in
secretagogue-induced hydrolysis of arachidonate from islet
phospholipids(31, 35, 36, 45, 50) ,
and, like an analagous myocardial enzyme(52) , islet
ASCI-PLA may be regulated by interaction with an isoform of
the glycolytic enzyme phosphofructokinase. A similar PLA has been reported to be activated by NO in a RAW 264.7 cells
because NO enhances arachidonate release from these cells; increasing
flux through glycolysis by increasing medium glucose concentration
increases NO-induced arachidonate release; and this effect is prevented
by the ASCI-PLA suicide substrate HELSS(55) .
Although incubation of islets with IL-1 did not increase expression of
islet ASCI-PLA activity in broken cell assays, pretreatment
of islets with HELSS did suppress IL-1-induced release of arachidonate
from islets, suggesting that ASCI-PLA may play at least a
permissive role in this phenomenon. In contrast to the case for RAW
264.7 cells, however, increasing medium glucose concentration did not
enhance IL-1-induced NO-dependent release of arachidonate from islets.
The difference between these two systems may be related to the fact
that NO inhibits islet phosphofructokinase activity (83) and
that IL-1 induces a specific decline in islet mRNA for the muscle
isoform of phosphofructokinase. ()H]arachidonate into lipids, and this
effect was prevented by the NO synthase inhibitor NMMA. When islets
were prelabeled with [H]arachidonate,
subsequent incubation with IL-1 also impaired retention of incorporated
radiolabel, and this effect was also prevented by NMMA. These
observations are consistent with the possibility that IL-1 suppresses
re-esterification of arachidonate into phospholipids through an
NO-dependent mechanism. That this mechanism may involve inhibition of
mitochondrial function by NO is suggested by the fact that the
mitochondrial ATP-synthase inhibitor oligomycin (57) also
suppressed esterification of [H]arachidonate into
islet phospholipids.. A HELSS-sensitive
Ca-independent PLA in P388D1 cells has
recently been reported to participate in the deacylation process
involved in remodeling of arachidonate-containing
phospholipids(85) . Ordinarily, deacylation of arachidonate is
rapidly followed by ATP-dependent conversion to arachidonoyl-CoA and
reacylation into phospholipids. The relative rapidity of reacylation
compared to deacylation in unstimulated cells maintains concentrations
of nonesterified arachidonate at low levels. IL-1-induced NO production
may reduce the reacylation rate and lead to accumulation of
nonesterified arachidonate. Blocking either the deacylation component
of the pathway with HELSS or preventing the NO-induced defect in
reacylation with NMMA abolishes effects of IL-1 to enhance accumulation
of nonesterified arachidonate in islets and to increase flux of this
substrate through the 12-lipoxygenase.
), prostaglandin E;
12-HETE, 12-hydroxy-(5,8,10,14)-eicosatetraenoic acid; 12-HPETE,
12-hydroperoxy-(5,8,10,14)-eicosatetraenoic acid; BSA, bovine serum
albumin; KRB, Krebs-Ringer bicarbonate buffer; IL-1,
interleukin-1; NMMA, N-monomethylarginine;
CHX, cycloheximide; NO, nitric oxide; iNOS, inducible nitric oxide
synthase; TMS, trimethylsilyl ether; PFBE, pentafluorobenzyl ester; GC,
gas chromatography; MS, mass spectrometry; NICI, negative ion chemical
ionization; HPLC, high performance liquid chromatography; RP, reverse
phase; NP, normal phase; PLA, phospholipase A;
TIIPLA, type II PLA; ASCI-PLA
,
ATP-stimulated, Ca-independent PLA;
SIN-1, 3-morpholinosydnonimine; RT, reverse transcriptase; PCR,
polymerase chain reaction: PBS, phosphate-buffered saline; HELSS, (E)-6-(bromomethylene)tetrahydro-3-(1-naphthal-enyl)-2H-pyran-2-one;
MEM, minimal essential medium; PBS, phosphate-buffered saline; bp, base
pair(s).)
The excellent technical assistance of Bingbing Li,
Zhi-Qin Hu, Dr. Mary Mueller, and Connie Marshall is gratefully
acknowledged. and that this message is not induced by IL-1.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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