©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Interleukin-1 Enhances Pancreatic Islet Arachidonic Acid 12-Lipoxygenase Product Generation by Increasing Substrate Availability through a Nitric Oxide-dependent Mechanism (*)

(Received for publication, June 13, 1995; and in revised form, October 19, 1995)

Zhongmin Ma (1) Sasanka Ramanadham (1) John A. Corbett (3) Alan Bohrer (1) Richard W. Gross (2) (4) (5) Michael L. McDaniel (3) John Turk (1)(§)

From the  (1)Mass Spectrometry Resource, Division of Endocrinology, Diabetes, and Metabolism, Division of Laboratory Medicine, (2)Division of Bioorganic Chemistry, Departments of Medicine, (3)Pathology, (4)Chemistry, and (5)Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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^G-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(2) (PLA(2)) activities nor mRNA levels for cytosolic or secretory PLA(2), a suicide substrate which inhibits an islet Ca-independent PLA(2) prevents enhancement of islet arachidonate release by IL-1. IL-1 also impairs esterification of [^3H(8)]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(2) activities. These results indicate that a novel action of NO is to increase levels of nonesterified arachidonic acid in islets.


INTRODUCTION

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(2)(^1)and the 12-lipoxygenase prod uct 12-hydroxy-(5,8,10,14)-eicosatetraenoic acid (12-HETE). PGE(2) 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(2) 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(2) are mediators which may adversely affect islet function(16, 17) .

Little is known about the influence of IL-1 on islet 12-HETE production, but 12-lipoxygenase products may promote insulin secretion from islet beta-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 beta-cells but not in alpha-cells or in pancreatic exocrine cells(18) . The beta-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.

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.


EXPERIMENTAL PROCEDURES

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-1beta was from Cistron Biotechnology (Pine Brook, NJ); N^G-monomethyl-L-arginine acetate from Calbiochem; actinomycin D from Sigma; and TranS-labeled methionine (1117 Ci/mmol) from ICN (Costa Mesa, CA). PGE(2) radioimmunoassay materials and [^2H(8)]12-HETE were from Cayman Chemical (Ann Arbor, MI). Both [^3H(8)]arachidonic acid and [^3H(8)]12-HETE (100 Ci/mmol) were obtained from DuPont NEN. [^2H(8)]Arachidonic acid was prepared by catalytic reduction of eicosa-(5,8,11,14)-tetrynoic acid (Hoffman LaRoche) with [^2H(2)]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(3), 5 mM KCl, 2.5 mM CaCl(2), 1 mM MgCl(2)); 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(2), 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 times 35 mm) and suspended in cCMRL medium (1 ml) containing no additives, IL-1beta (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(2), 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 times 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(2), 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 times 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(2) were extracted from medium and measured. Insulin content of islet pellets was determined as described (3) after extraction with 75% ethanol, 1.5% HCl.

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.

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 and Insulin Measurement

PGE(2) 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(2) 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, [^2H(8)]12-HETE (20 ng) and [^3H(8)]12-HETE (50 nCi) were added as internal standards, and medium was acidified (pH 3.0, 1 N HCl) and extracted twice (CH(2)Cl(2)). 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 times 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 (CH(2)Cl(2)), concentrated to dryness, converted to the trimethylsilyl ether (TMS) derivative, and analyzed by GC-NICI-MS. Quantitation of 12-HETE was performed relative to [^2H(8)]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, [^2H(8)]arachidonic acid (100 ng), and [^3H(8)]arachidonic acid (50 nCi) were added as internal standards. Medium was acidified (pH 3.0, 1 N HCl) and extracted twice (CH(2)Cl(2)). 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 (CH(2)Cl(2)), concentrated to dryness, converted to a PFBE derivative, and analyzed by GC-NICI-MS. Quantitation of arachidonate was performed relative to [^2H(8)]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, CH(2)Cl(2) (200 µl) added and vortex-mixed, and aqueous phase discarded. The PFBE derivative in the CH(2)Cl(2) 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 [O]12-HETE

Racemic [^18O(2)]12-HETE was prepared from unlabeled, racemic 12-HETE (BIOMOL, Philadelphia, PA) by butyrylcholinesterase (Type XI, Sigma)-catalyzed exchange in H(2)^18O and purified by RP-HPLC (8, 9) . The blank value ([O(2)]/[^18O(2)] 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 [^18O(2)]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 (CH(2)Cl(2)), concentrated to dryness, reconstituted in mobile phase, and analyzed by chiral phase HPLC with two Baker-Bond (dinitrobenzoylphenylglycine) ionically coupled columns (4.3 times 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 [^18O(2)]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^2). 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 [^18O(2)]12-HETE-(PFBE, TMS), 399 for [^2H(8)]12-HETE-(PFBE, TMS), 303 for endogenous arachidonate-PFBE, and 311 for [^2H(8)] 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-1beta (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(3)) 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 times 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 times 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% beta-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) .

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(2). Islets were isolated (centrifugation, 5000 times 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 H(2)O (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(2); 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(2)(43) , sense 5`-TGTTCAACAGAGTTTTGG and antisense 5`-AACAGAGCAACGAGATGG (expected fragment 983 bp); 14-kDa type II PLA(2)(44) , sense 5`-AGTTTGGGCAAATGATTCTG and antisense 5`-TCTTTCAGCAACTGGGCGTC (expected fragment 372 bp).


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(2) (cPLA(2), third set of 3 lanes), or a 14-kDa type II PLA(2) (TIIPLA(2), 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(2) or TIIPLA(2) 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 times g, 10 min). Mitochondrial pellets obtained after further centrifugation (10,000 times g, 10 min) were discarded and resultant supernatants centrifuged (170,000 times g, 60 min) to yield membranous pellets and cytosolic supernatants. Membranous fractions were resuspended in homogenization buffer.

Phospholipase A Activity Measurements

PLA(2) 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(2)). In experiments examining effects of NMMA (0.5 mM) or SIN-1 (0.1 mM) on PLA(2) activity, test compounds were added 5 min before addition of substrate. In experiments examining effects of prior incubation with IL-1 on islet PLA(2) 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-[^3H(2)])-octadec-9`-enoyl-sn-glycero-3-phosphoc holine or 1-hexadecanoyl-2-(1-[^14C])-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 times 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(F) 0.58) from diglycerides (R(F) 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(2) 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 times D times 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 [^3H] (about 0.4) or [^14C] (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 [H]Arachidonic Acid into Islet Lipids

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 times 35 mm) to which were added 10 µCi (for IL-1 experiments) or 0.5 µCi (for oligomycin experiments) of [^3H(8)]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 ^3H-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 times 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). ^3H-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 [^3H(8)]Arachidonic Acid in Islet Lipids

Islets were first prelabeled with [^3H(8)]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 times 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 ^3H-content determined by liquid scintillation spectrometry.

Reverse Phase HPLC Analysis of [^3H]Arachidonate Released from Prelabeled Islets Treated with Interleukin-1 and Other Agents

In experiments described in Table 1, [^3H]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 (CH(2)Cl(2)), concentrated to dryness, reconstituted in CH(3)OH (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 ^3H-content of the arachidonate peak (retention volume about 50 ml) was determined by liquid scintillation spectrometry.




RESULTS

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(2) and insulin by immunoassay. As illustrated in Fig. 1, 17 mMD-glucose plus carbachol stimulated insulin secretion and production of PGE(2) and 12-HETE by control islets, and prior IL-1 exposure augmented PGE(2) 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(2) produced under these conditions was released into medium, and little remained associated with islets (not shown).


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(2) (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(2) 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.



The time course of production of 12-HETE and PGE(2) 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(2), 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(2), 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.


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(2) (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(2), 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.



The effects of IL-1 to induce an increase in islet PGE(2) 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(2) 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(2) 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.


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(2) (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(2), 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.



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 [^18O(2)]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.


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 [^18O(2)]12-HETE internal standard, converted to the PFBE derivative, purified by reverse phase HPLC, and analyzed by chiral phase HPLC. [^18O(2)]12-HETE rather than [^2H(8)]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 [^18O(2)]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(2)]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.



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(2) 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(2) (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).


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(2) (panel A) and 12-HETE (panel B) were quantitated as described in the legend to Fig. 2. Values for PGE(2) 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.



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(2) (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.


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(2) (panel C) by immunoassay. Values for arachidonate represent absolute mass (ng). Values for 12-HETE and PGE(2) 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(2) were 1009, 34.6, and 133.2 pmol, respectively, and mean acid-extractable insulin content of islet pellets was 14.3 nmol.



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-dependent or Ca-independent PLA(2) activities in islet cytosolic or membranous fractions (not shown). This suggests that islet PLA(2) enzymes are neither directly inhibited by NMMA nor directly activated by NO.


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(2) enzymes, both Ca-dependent and Ca-independent PLA(2) 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(2) 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(2) (cPLA(2)), for a type II 14-kDa PLA(2) (TIIPLA(2)), 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(2), TIIPLA(2), 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(2) or TIIPLA(2).

In addition to cPLA(2) and TIIPLA(2), a third PLA(2) activity expressed in islets is an ATP-stimulated, Ca-independent (ASCI)-PLA(2)(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(2) is activated by a regulatory subunit which is an isoform of the glycolytic enzyme phosphofructokinase(52) . Both islet and myocardial ASCI-PLA(2) are inhibited by a haloenol lactone suicide substrate (HELSS) which does not inhibit cPLA(2) or TIIPLA(2)(53, 54) . NO has recently been reported to activate an ASCI-PLA(2) in RAW 264.7 cells because NO augments release of [^3H]arachidonic acid from prelabeled cells; this effect is enhanced by increasing medium glucose concentration; and the effect is suppressed by HELSS(55) . ASCI-PLA(2) 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) .

To examine the possibility that ASCI-PLA(2) 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 [^3H(8)]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 [^3H]arachidonic acid was measured by RP-HPLC. At 5 mM glucose, IL-1 induced more than a doubling of [^3H]arachidonic acid release, and this effect was prevented by NMMA (Table 1, panel A). IL-1-induced release of [^3H]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 [^3H]arachidonic acid release. Pretreatment of islets with the ASCI-PLA(2) suicide substrate HELSS prevented IL-1-induced enhancement of [^3H]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(2) 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(2) 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(2) enzymes.

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 [^3H(8)]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). [^3H(8)]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 [^3H(8)]arachidonate and then incubated for 24 h with no additions, with IL-1 alone, or with IL-1 plus NMMA, retention of [^3H]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 [^3H(8)]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).


Figure 11: Influence of prior incubation with interleukin-1 with or without NMMA on incorporation of [^3H]arachidonic acid into islet lipids and on retention of previously incorporated [^3H]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 [^3H(8)]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 ^3H-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 ^3H-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 ^3H-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 [^3H(8)]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 ^3H-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 ^3H-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 ^3H-lipid content of control islets compared to that of islets that had been incubated with IL-1 + NMMA.



The magnitude of the effect of prior IL-1-treatment to suppress esterification of [^3H]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 [^3H]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 [^3H]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 [^3H]arachidonic acid into islet lipids (Fig. 14).


Figure 12: Influence of prior incubation of interleukin-1 with or without NMMA on the time course of incorporation of [^3H] 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 [^3H(8)]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 ^3H-content determined. Error bars indicate S.E. (n = 3).




Figure 13: Normal phase HPLC analysis of [^3H]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 [^3H(8)]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 ^3H-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).




Figure 14: Influence of oligomycin on incorporation of [^3H] 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 [^3H(8)]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 ^3H-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.




DISCUSSION

Interleukin-1 impairs insulin secretion by pancreatic islets and induces islet expression of cyclooxygenase and NO synthase, resulting in overproduction of PGE(2) 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.

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(2) 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(2) (cPLA(2)) participates in arachidonate release in some cells (63, 64, 65, 66, 67) and is expressed in human islets(68) . Immunochemical evidence indicates that cPLA(2) is expressed at very low levels in rat islets(69) . Our RT-PCR data indicate that cPLA(2) mRNA is expressed in rat islets but is not induced by IL-1. Low molecular mass (14 kDa) Ca-dependent PLA(2) enzymes also exist in islets(70) . Our R T-PCR data indicate that islets express mRNA for a 14-kDa type II PLA(2), but IL-1 treatment does not increase its abundance. Activity measurements also failed to demonstrate increased expression of Ca-dependent PLA(2) 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(2) by reversible phosphorylation (67, 81) or by NO-amplified Ca entry (82) might also not be demonstrable with broken cell assays.

Islets express an ATP-stimulated, Ca-independent PLA(2) (ASCI-PLA(2)) 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(2) may be regulated by interaction with an isoform of the glycolytic enzyme phosphofructokinase. A similar PLA(2) 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(2) suicide substrate HELSS(55) . Although incubation of islets with IL-1 did not increase expression of islet ASCI-PLA(2) activity in broken cell assays, pretreatment of islets with HELSS did suppress IL-1-induced release of arachidonate from islets, suggesting that ASCI-PLA(2) 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. (^2)

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 [^3H(8)]arachidonate into lipids, and this effect was prevented by the NO synthase inhibitor NMMA. When islets were prelabeled with [^3H(8)]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 [^3H]arachidonate into islet phospholipids.

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(2). A HELSS-sensitive Ca-independent PLA(2) 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.

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.


FOOTNOTES

*
This research was supported by a grant from the Juvenile Diabetes Foundation International(193192) and by National Institutes of Health Grants R37-DK-34388 and P41-RR-00954. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Mass Spectrometry Resource, Division of Diabetes, Endocrinology, and Metabolism, Box 8127, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110.

(^1)
The abbreviations used are: PGE(2), prostaglandin E(2); 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-1beta; NMMA, N^G-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(2), phospholipase A(2); TIIPLA(2), type II PLA(2); ASCI-PLA(2), ATP-stimulated, Ca-independent PLA(2); 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).

(^2)
Z. Ma and J. Turk, manuscript in preparation.


ACKNOWLEDGEMENTS

The excellent technical assistance of Bingbing Li, Zhi-Qin Hu, Dr. Mary Mueller, and Connie Marshall is gratefully acknowledged.

Note Added in Proof-We have also found that rat pancreatic islets express mRNA for a 14-kDa type I PLA(2) and that this message is not induced by IL-1.


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