Glucose homeostasis, insulin secretion, and islet phospholipids in mice

7/27/2019 Glucose homeostasis, insulin secretion, and islet phospholipids in mice

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doi:10.1152/ajpendo.00474.2007294:E217-E229, 2008. First published 25 September 2007;Am J Physiol Endocrinol Metab

Louis H. Philipson and John TurkShunzhong Bao, David A. Jacobson, Mary Wohltmann, Alan Bohrer, Wu Jin,

-null mice2-cells and in iPLApancreaticin2phospholipids in mice that overexpress iPLA

Glucose homeostasis, insulin secretion, and islet

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Glucose homeostasis, insulin secretion, and islet phospholipids in mice that

overexpress iPLA2 in pancreatic -cells and in iPLA2-null mice

Shunzhong Bao,1 David A. Jacobson,2 Mary Wohltmann,1 Alan Bohrer,1 Wu Jin,1 Louis H. Philipson,2

and John Turk1

1Division of Endocrinology, Metabolism, and Lipid Research, Department of Medicine, Washington University Schoolof Medicine, St. Louis, Missouri; and 2Department of Medicine, University of Chicago, Chicago, Illinois

Submitted 23 July 2007; accepted in final form 23 September 2007

Bao S, Jacobson DA, Wohltmann M, Bohrer A, Jin W,Philipson LH, Turk J. Glucose homeostasis, insulin secretion,and islet phospholipids in mice that overexpress iPLA2 in pan-creatic -cells and in iPLA2-null mice. Am J Physiol Endocrinol

Metab 294: E217E229, 2008. First published September 27,2007; doi:10.1152/ajpendo.00474.2007.Studies with geneticallymodified insulinoma cells suggest that group VIA phospholipase A2(iPLA2) participates in amplifying glucose-induced insulin secre-tion. INS-1 insulinoma cells that overexpress iPLA2, for example,exhibit amplified insulin-secretory responses to glucose and cAMP-elevating agents. To determine whether similar effects occur in wholeanimals, we prepared transgenic (TG) mice in which the rat insulin 1promoter (RIP) drives iPLA2 overexpression, and two characterizedTG mouse lines exhibit similar phenotypes. Their pancreatic isletiPLA2 expression is increased severalfold, as reflected by quantita-tive PCR of iPLA2 mRNA, immunoblotting of iPLA2 protein, andiPLA2 enzymatic activity. Immunofluorescence microscopic studiesof pancreatic sections confirm iPLA2 overexpression in RIP-iPLA2-TG islet -cells without obviously perturbed islet morphol-ogy. Male RIP-iPLA2-TG mice exhibit lower blood glucose andhigher plasma insulin concentrations than wild-type (WT) mice whenfasting and develop lower blood glucose levels in glucose tolerancetests, but WT and TG blood glucose levels do not differ in insulintolerance tests. Islets from male RIP-iPLA2-TG mice exhibit greater

amplification of glucose-induced insulin secretion by a cAMP-elevat-ing agent than WT islets. In contrast, islets from male iPLA2-nullmice exhibit blunted insulin secretion, and those mice have impairedglucose tolerance. Arachidonate incorporation into and the phospho-lipid composition of RIP-iPLA2-TG islets are normal, but theyexhibit reduced Kv2.1 delayed rectifier current and prolonged glu-cose-induced action potentials and elevations of cytosolic Ca2 con-centration that suggest a molecular mechanism for the physiologicalrole of iPLA2 to amplify insulin secretion.

transgenic mice; glucose tolerance; insulin tolerance

GLUCOSE HOMEOSTASIS REQUIRES that pancreatic -cells secreteinsulin when blood glucose concentrations exceed 5 mM.

Autoimmune -cell destruction causes type 1 diabetes melli-tus, and in type 2 diabetes mellitus, there is 50% loss of-cellmass and impaired insulin secretion and action (12, 15). Inten-sive insulin therapy reduces diabetic complications but in-creases risks of hypoglycemia (16), and -cell replacementmight someday be a superior therapy (59, 62).

Understanding control of-cell growth, death, and secretionmight permit iterative introduction of genes into -cell lines orprecursors to optimize secretion, proliferation, and resistance

to injury (24, 69). Combined with empirical selection andencapsulation, such -cell engineering might someday providea renewable source of-cells for replacement therapy (44, 45).Developing such potential future therapies requires increasedunderstanding of the genes and gene products that govern-cell physiology (46).

Insulin secretion involves glucose transport into -cells,phosphorylation by glucokinase at a rate proportional to extra-cellular glucose concentrations, and further metabolism that

increases the ATP/ADP concentration ratio (20, 39, 40). Thisinactivates -cell ATP-sensitive K channels (KATP), causingmembrane depolarization, voltage-operated Ca2-channel opening,and a rise in Ca2 concentration ([Ca2]) that induces exocy-tosis (1, 14, 21). Several other, incompletely understood pro-cesses modulate or amplify insulin secretion, e.g., nonselectivecation channels activated by Ca2-store depletion, an electrogenicNa-K-ATPase, and voltage-operated K channels also affect -cellmembrane potential and secretion (17, 23, 29, 48).

Reducing equivalent shuttles to and from mitochondria arealso required for secretion (19, 58, 76), and glucose augmentssecretion by KATP-independent mechanisms (60, 77). Elevating-cell cAMP also amplifies secretion by protein kinase A (PKA)-dependent and -independent mechanisms (50, 61), and -cellsignaling involves phospholipid hydrolysis and accumulation ofphospholipid-derived mediators (31, 41, 48, 50, 52, 63, 68, 70).

We and others (48, 68, 70) have developed evidence that anislet phospholipase A2 that does not require Ca

2 (iPLA2) isactivated by secretagogues and that its products participate in-cell signaling. Characterizing this activity resulted in ourcloning an 84-kDa phospholipase A2 from islet cDNA libraries(34, 37). Recombinant iPLA2 is Ca

2 independent, activatedby ATP, and inhibited by a suicide substrate that also attenu-ates glucose-induced insulin secretion, arachidonate release,and rises in -cell [Ca2] (52).

Genetic gain- and loss-of-function studies with insulinomacells support the possible involvement of iPLA2 in insulin

secretion. INS-1 insulinoma cells stably transfected to expressshort interfering RNA (siRNA) that reduces iPLA2 expres-sion exhibit reduced insulin secretion when stimulated withglucose and markedly impaired amplification of insulin secre-tion by cAMP-elevating agents (5). In contrast, stably trans-fected INS-1 cells that overexpress iPLA2 exhibit amplifiedinsulin-secretory responses to glucose, particularly in the presenceof cAMP-elevating agents, and this is associated with subcellularredistribution and membrane association of iPLA2 (35).

Address for reprint requests and other correspondence: J. Turk, WashingtonUniv. School of Medicine, Box 8127, 660 S. Euclid Ave., St. Louis, MO 63110(e-mail: [email protected]).

The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Am J Physiol Endocrinol Metab 294: E217E229, 2008.First published September 27, 2007; doi:10.1152/ajpendo.00474.2007.

0193-1849/08 $8.00 Copyright 2008 the American Physiological Societyhttp://www.ajpendo.org E217


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To determine whether these findings with cultured insulin-oma cells are relevant to animal physiology, we have preparedtransgenic (TG) mice in which iPLA2 overexpression isdriven by the rat insulin 1 promoter (RIP) and studied fastingconcentrations of glucose and insulin, blood glucose excur-sions on administration of glucose or exogenous insulin, andinsulin-secretory and electrophysiological responses of pancre-

atic islets isolated from RIP-iPLA2-TG and wild-type (WT)mice.

EXPERIMENTAL PROCEDURES

Materials. (E)-6-(bromo-methylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (BEL) was obtained from Cayman Chemical (AnnArbor, MI); enhanced chemiluminescence reagents from AmershamBiosciences (Piscataway, NJ); SDS-PAGE supplies from Bio-Rad(Richmond, CA); ATP, common reagents, and salts from Sigma (St.Louis, MO); culture media, penicillin, streptomycin, Hanks balancedsalt solution, L-glutamine, agarose, molecular mass standards, andRT-PCR reagents from Invitrogen (Carlsbad, CA); fetal bovine serumfrom Hyclone (Logan, UT); Pentex bovine serum albumin (BSA, fattyacid-free, fraction V) from ICN Biomedical (Aurora, OH); and fors-kolin from Calbiochem (La Jolla, CA). Krebs-Ringer bicarbonate(KRB) buffer contained (in mM) 25 HEPES (pH 7.4), 115 NaCl, 24NaHCO3, 5 KCl, 1 MgCl2, and 2.5 CaCl2.

Generation and genotyping of RIP-iPLA2-TG mice. As describedpreviously (10), full-length iPLA2 cDNA was inserted in a RIP-I/-globin expression vector and was microinjected into fertilized eggsof C57BL/6J mice. TG founders were mated with WT C57BL/6J mice(Jackson Laboratory), and N2 and N3 generations from two lines withsimilar phenotypes were studied. The genetic background of resultantmice was pure C57BL/6J, and WT littermates were used as controls.All protocols were approved by the Washington University AnimalStudies Committee.

Genotyping was performed with tail-clipping DNA either bySouthern blotting analyses or by PCR. For Southern blots, DNA wasdigested with EcoR I restriction endonuclease. Digests were analyzed

by electrophoresis and were transferred to nylon membranes, whichwere incubated with a 32P-labeled probe that recognizes the sequencein the rabbit hemoglobin gene contained in the original construct. ForPCR analyses, DNA was used as a template with two pairs of primers.One pair amplified the sequence in the internal control fatty acid-binding protein gene (Fabpi) gene, and the primer sequences were(Fabpi 5) CCT CCG GAG AGC AGC GAT TAA AAG TGT CAGand (Fabpi 3) TAG AGC TTT GCC ACA TCA CAG GTC ATTCAG. The expected size of the product was 450 bp. The other primerpair amplified the sequence that spans the junction of iPLA2 andglobin cDNA in the TG construct. The primer sequences were (TG 5)cta ggc tca gac atc atg ctg gac gag gt and (TG 3 ) aag atc tca gtg gtattt gtg agc cag gg. The expected size of the product was 200 bp.

Generating and genotyping iPLA2-null mice. Preparation of theiPLA2 knockout construct, its introduction into 129/SvJ mouse

embryonic stem cells, their selection with G418, characterization bySouthern blotting, injection into C57BL/6 mouse blastocysts, theproduction of chimeras and then heterozygotes, and the mating ofheterozygotes to yield WT, heterozygous, and iPLA2-null mice in aMendelian distribution are described elsewhere, as is their genotypingby Southern blotting of tail genomic DNA (79). The genetic back-ground of the resultant mice is mixed 129/SvJ C57BL/6.

Islet isolation. Islets were isolated from pancreata of male WT,RIP-iPLA2-TG, and iPLA2-null mice by collagenase digestionafter mincing, followed by Ficoll step density gradient separation andmanual selection under stereomicroscopic visualization to excludecontaminating tissues (9, 49). Mouse islets were counted and used forPCR and immunoblotting of iPLA2 mRNA and protein, respec-tively; for measuring iPLA2-specific enzymatic activity and ex vivo

secretion of insulin and electrophysiological responses; and for ex-traction of phospholipids.

PCR of iPLA2 mRNA in mouse islets. As described previously (9,13), total RNA was extracted with TRIzol reagent (Invitrogen). Aftertreatment with DNase I, 1 g of total RNA was reverse transcribedwith an oligo(dT) primer, and quantitative PCR was performed withan ABI Prism 7700 PCR instrument (Applied Biosystems) using theSYBR Advantage qPCR Premix (Clontech). Each assay included a

negative control using RNA not subjected to reverse transcription.PCR was performed with a pair of primers designed to amplify afragment of iPLA2 cDNA. The sequence of primer 1 was GCC CTGGCC ATT CTA CAC AGT A, and that of primer 2 was CAC CTCATC CTT CAT ACG GAA GT. Amplification specificity was verifiedby agarose gel electrophoresis of products and a heat-dissociationprotocol. Sequence-specific amplification was detected with increas-ing fluorescent signal of FAM (reporter dye) during the amplificationcycle. The amplification of 18s rRNA served as control.

Western-blotting analyses. As described previously (35), proteinsin islet homogenates were analyzed by SDS-PAGE (7.5%), trans-ferred onto a PVDF membrane, and probed with antibody 506provided by Dr. Richard Gross (Washington University, St. Louis,MO). Protein bands were visualized by ECL. Membranes were alsoprobed with actin antibodies as a loading control.

Ca2-independent PLA2 activity assay. As described previously(37), the protein content of islet cytosolic fractions was determined byBio-Rad assay, and iPLA2 activity was measured in aliquots (20 gprotein) added to assay buffer (200 mM Tris HCl, pH 7.0; total assayvolume, 200 l) containing 5 mM EGTA with or without 1 mM ATP.Some aliquots were pretreated (2 min) with BEL (10 M) beforeassay. Reactions were initiated by injecting substrate (L--1-palmi-toyl-2-[14C]arachidonoyl-phosphatidylethanolamine; specific activity,50 Ci/mol; final concentration, 5 M) in ethanol (5 l). Assaymixtures were incubated (3 min, 37C), and reactions were terminatedby adding butanol (0.1 ml) and vortexing. After centrifugation (2,000g, 4 min), products in the butanol layer were analyzed by silica gel GTLC in hexane/ethyl ether/acetic acid (80:20:1). The TLC regioncontaining free arachidonic acid (Rf, 0.58) was scraped into vials, andits 14C content was determined. Specific activity was calculated from

released 14C dpm and protein content.Extracting islet phospholipids. As described previously (9), islets

were placed in a solution (2 ml) of chloroform/methanol (1/1, vol/vol), homogenized, and sonicated on ice (20% power, 5-s bursts for60 s; Vibra Cell probe sonicator; Sonics and Materials, Danbury, CT).After centrifugation (2,800 g, 5 min) to remove tissue debris, super-natants were transferred to silanized 10-ml glass tubes and wereextracted with methanol (1 ml), chloroform (1 ml), and water (1.8 ml).Samples were vortex mixed and centrifuged (900 g, 5 min). Super-natants were removed, concentrated, and dissolved in methanol/chloroform (9/1), and lipid phosphorus content was determined.

Immunofluorescence microscopy. As described previously (10),pancreata were removed from mice, fixed in 4% paraformaldehyde,and embedded in paraffin to prepare sections, which were placed onglass slides and deparaffinized. Immunostaining for insulin was

performed with guinea pig anti-human insulin antibody (1:300;BioGenex Laboratories, San Ramon, CA) and FITC-conjugated sec-ondary antibody. Immunostaining for glucagon was performed withrabbit anti-glucagon (1:500; Chemicon International, Temecula, CA)as the primary antibody. Incubations with primary antibodies wereperformed overnight in a humidified chamber. Secondary antibodieswere donkey anti-guinea pig FITC (for insulin) and donkey anti-rabbitCy3 (for glucagon). Slides were examined with a Nikon TE300microscope.

Fasting and fed blood glucose and insulin concentrations. Asdescribed previously (10), blood samples were obtained from the lateralsaphenous vein in heparinized capillary tubes, and glucose concentra-tions were measured in whole blood with a blood-glucose monitor(Becton Dickenson) or an Ascensia ELITE XL blood-glucose meter.

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Plasma was prepared from heparinized blood by centrifugation, andinsulin levels were determined in aliquots (5 l) with a rat insulinELISA kit (Crystal Chem). Fasting blood samples were obtained afteran overnight fast, and fed blood samples were obtained between 9:00and 10:00 AM.

Glucose and insulin tolerance tests. As described (9), intraperito-neal glucose tolerance tests (GTT) were performed in mice fastedovernight from which a baseline blood sample was obtained, followedby intraperitoneal injection of D-glucose (2 mg/g) and collection ofblood for measurement of glucose after 30, 60, and 120 min. OralGTT (42) were performed in a similar manner except that glucose(2 mg/kg) was administered enterally by gavage. Insulin tolerancetests were performed in mice with free access to water and chow thatreceived an intraperitoneal injection (0.75 U/kg) of human regularinsulin (Lilly, Indianapolis, IN), followed by collection of blood after30, 60, and 120 min for glucose determinations (9, 49).

Insulin secretion by isolated pancreatic islets. Islets were isolatedfrom pancreata of male mice as described in Islet isolation. Insulin-secretion studies were performed with 30 islets per incubation, asdescribed previously (9, 49). Islets were rinsed with KRB mediumcontaining 3 mM glucose and 0.1% BSA and were placed in silanizedtubes (12 75 mm) in the same buffer, through which 95% air-5%

CO2

was bubbled before incubations. Tubes were capped and incu-bated (37C, 30 min) in a shaking water bath. Buffer was thenreplaced with KRB medium containing 3, 8, or 20 mM glucose and0.1% BSA without or with forskolin (2.5 M), and samples wereincubated for 30 min. Secreted insulin was measured by radioimmu-noassay.

Determination of [3H]arachidonic acid incorporation into islet

phospholipids. As described earlier (55), islets were washed thrice inKRB medium containing 5.5 mM glucose and 0.1% BSA, resus-pended in that medium, and preincubated for 30 min at 37C. Islets(100 per condition) were then placed in fresh KRB medium thatcontained 5.5 mM glucose, 0.1% BSA, and 2.5 mM CaCl2, and[3H]arachidonic acid (final concentration 0.5 Ci/ml, 5 nM) wasadded to the medium. Incubations were performed for 1060 min at37C, and islets were then washed thrice in KRB medium containing 5.5

mM glucose and 0.1% BSA to remove unincorporated [3

H]arachidonicacid. Phospholipids were then extracted as described above and wereanalyzed by TLC, and the [3H]arachidonate content of glycerophos-phocholine (GPC) lipids was determined by liquid scintillation spec-trometry and normalized to lipid phosphorus content.

Positive ion electrospray ionization mass spectrometric analyses of

choline-containing glycerolipids. Diradyl-GPC lipids and lysophos-phatidylcholine (LPC) species were analyzed as Li adducts bypositive ion electrospray ionization mass spectrometric analyses (ESI/MS) on a Finnigan (San Jose, CA) TSQ-7000 triple-stage quadrupolemass spectrometer with an ESI source controlled by Finnigan ICISsoftware. Phospholipids were dissolved in methanol/chloroform (2/1,vol/vol) containing LiOH (10 pmol/l), infused (1 l/min) with aHarvard syringe pump, and analyzed as described (25, 26). Fortandem MS, precursor ions selected in the first quadrupole were

accelerated (3236 eV collision energy) into a chamber containingargon (2.32.5 mTorr) to induce collision-activated dissociation, andproduct ions were analyzed in the final quadrupole. Identities of GPCspecies were determined from their tandem spectra (25, 26), and theirquantities were determined relative to internal standards by interpo-lation from standard curves (5, 55).

Whole cell ruptured-patch electrophysiological recording. Volt-age-activated currents were recorded by using whole cell ruptured-patch clamps with an Axopatch 200B amplifier and pCLAMP 9software (Molecular Devices), as described (28, 29). Patch electrodes(24 M) were loaded with intracellular solution containing (inmmol/l) 140 KCl, 1 MgCl2[H2O]6, 10 EGTA, 10 HEPES, 5 MgATP(pH 7.25) with KOH. Islets were perfused with KRB containing (inmmol/l) 119 NaCl, 2 CaCl2[(H2O)6], 4.7 KCl, 10 HEPES, 1.2

MgSO4, 1.2 KH2PO4, adjusted to pH 7.3 with NaOH and with theindicated glucose concentration.

Islet perforated-patch electrophysiological recording. Patch elec-trodes (24 M) were loaded with intracellular solution containing(in mmol/l) 140 KCl, 1 MgCl2[H2O]6, 10 EGTA, 10 HEPES (pH7.25) with KOH containing the pore-forming antibiotic amphotericinB (52) (Sigma), as described (28, 29). Islets were perfused with KRBcontaining (in mmol/l) 119 NaCl, 2 CaCl2[(H2O)6], 4.7 KCl, 10

HEPES, 1.2 MgSO4, 1.2 KH2PO4, and glucose as indicated, adjustedto pH 7.3 with NaOH. Cells on the periphery of islets on glasscoverslips were sealed in voltage clamp at 80 V, and the ampho-tericin allowed perforation and good access over several minutes.After being switched to current clamp, cells that had a restingmembrane voltage near 65 mV in 2 mM glucose and near 75 mVin 0 mM glucose were assumed to be -cells. The glucose-containingsolution (at 37C) was perfused into a heated chamber (at 37C).

High-speed intracellular calcium imaging. Mouse islets attached toglass-bottom Matek tissue-culture dishes were loaded with 5 M fluo4-AM loaded in Krebs-Ringer-Henseleit 2 buffer for 30 min at 37C,as described (28, 29). Loaded islets were placed in a temperaturecontroller (TC-202; Medical Systems) mounted on the stage of aninverted microscope (Nikon Eclipse TE2000-U) for imaging. Exper-iments were performed at 37C. Islets were excited at 488 nm from a

monochrometer (Till Photonics), and the emitted light was filteredwith a 535/40 filter (Chroma) and recorded with a photomultipliertube (Photon Technology International). Acquisition was controlled

Fig. 1. Preparation of transgenic (TG) mice that overexpress group VIAphospholipase A2 (iPLA2) in pancreatic islet -cells. A: construct used toprepare TG mice. Full-length iPLA2 cDNA was inserted into the rat insulinpromoter (RIP)-I/-globin (G) expression vector downstream of RIP andwithin -globin gene sequence. B: Southern blot of tail-clipping DNA fromwild-type (W) or RIP-iPLA2-TG mice of line TG1 or TG2 with a 32P-labeledprobe that recognizes transgene sequence. C: PCR analyses using DNA fromwild-type (W) or RIP-iPLA2-TG (T) mice, and primers that amplify sequencethat either spans junction between iPLA2 and globin cDNA (200-bp product,lower band) or is within internal control Fabpi (450-bp product, upper band).

Lane M represents molecular weight markers and lane N a control reactionwithout template.

E219GLUCOSE HOMEOSTASIS IN iPLA2-OVEREXPRESSING MICE



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with pCLAMP 9 software. In all experiments, to obtain data withsufficiently high time resolution, we used 10-kHz acquisition. Dataanalysis was performed with pCLAMP 9 and Microsoft Excel.

Statistical methods. Results are presented as means SE. Datawere evaluated by unpaired, two-tailed Students t-test or by analysisof variance with appropriate post hoc tests. Significance levels aredescribed in figure legends.

RESULTS

Generation of TG mice that overexpress iPLA2 in pancre-atic islet-cells. In the construct used to generate the TG mice(Fig. 1A), rat iPLA2 cDNA was inserted downstream of theRIP at a site within the rabbit globin gene sequence. Transcrip-tion of the sequence encoding iPLA2 is under control of RIP,and TG overexpression of iPLA2 is expected in cells thatexpress insulin, e.g., pancreatic islet -cells, but not in othercells. Transgene incorporation was determined by Southernblotting (Fig. 1B) and PCR (Fig. 1C) analyses. Two founderswere identified, and progeny from each were viable and fertile.Mice from TG lines TG1 and TG2 exhibited similar pheno-types.

Overexpression of iPLA2 in pancreatic islet-cells of TGmice. Real-time PCR measurements indicated that iPLA2mRNA was expressed in islets isolated from RIP-iPLA2-TGmice at levels severalfold higher than those from WT littermates(Fig. 2A). Immunoblotting analyses similarly indicated thatiPLA2 protein is expressed at much higher levels in isletsfrom RIP-iPLA2-TG compared with WT mice (Fig. 2B). TheiPLA2 enzymatic-specific activity was 12-fold higher in isletsfrom RIP-iPLA2-TG mice compared with those from WTmice, and activity was stimulated by ATP and inhibited by thesuicide substrate BEL (Fig. 2C), as is characteristic of iPLA2(8, 9, 3437). The size and morphology of pancreatic isletsfrom RIP-iPLA2-TG mice were not obviously different from

those of WT mice, but iPLA2 expression, as visualized byfluorescence microscopy with antibodies to iPLA2, was muchgreater in islets from RIP-iPLA2-TG mice (Fig. 3).

Growth of RIP-iPLA2-TG male mice. The growth, devel-opment, and food intake of RIP-iPLA2-TG and WT malemice did not differ appreciably, and Fig. 4 illustrates that therates of weight gain of WT and RIP-iPLA2-TG male mice onan ad libitum regular chow diet were not statistically distin-guishable between the ages of 3 and 48 wk.

Glucose homeostasis in RIP-iPLA2-TG male mice. In the

fasting state, blood glucose concentrations were significantly lowerin RIP-iPLA2-TG male mice than in WT mice (Fig. 5A), andfasting insulin levels were significantly higher in RIP-iPLA2-TG male mice (Fig. 5B). After oral glucose adminis-tration, blood glucose levels were significantly lower in RIP-iPLA2-TG male mice compared with WT mice at 15 min andremained somewhat but not significantly lower at 30, 60, or120 min (Fig. 6A). Although the fractional increase in peakglucose levels relative to the baseline fasting value was similarfor WT (2.51 0.11-fold) and RIP-iPLA2-TG (2.55 0.13-fold) mice, the area under the concentration vs. time curve(AUC) of the GTT was significantly (1.17 0.05-fold) greaterfor WT [434 19 arbitrary units (AU)] than for RIP-

iPLA2-TG (370 12 AU) mice. Baseline insulin levels weresignificantly higher in RIP-iPLA2-TG male mice than in WTmice, and insulin levels in both groups rose somewhat less thantwofold at 15 min after oral glucose administration (Fig. 6B).

After intraperitoneal administration of glucose, blood glu-cose concentrations at 30, 60, and 120 min were all signifi-cantly lower in RIP-iPLA2-TG male mice than in WT mice(Fig. 7A), and the AUC for the GTT was also significantly(1.18 0.05-fold) greater for WT (605 25 AU) than forRIP-iPLA2-TG (514 20 AU) mice. Blood glucose concen-trations were similar in the two groups at all tested time pointsafter intraperitoneal administration of insulin (Fig. 7B). Thissuggests that sensitivity to insulin is not altered in RIP-

iPLA2-TG male mice, which in turn suggests that the lowerglucose levels observed in GTT might reflect enhanced insulinsecretion in RIP-iPLA2-TG male mice. To evaluate this

Fig. 2. Overexpression of iPLA2 in pancre-atic islets of RIP-iPLA2-TG mice. A: real-time PCR analyses of iPLA2 mRNA levelsin islets from wild-type (WT) or RIP-iPLA2-TG mice of l ine TG1 or TG2.

B: Western blot of immunoreactive iPLA2protein in pancreatic islets isolated from WTor RIP-iPLA2-TG mice of line TG1 or TG2.

Immunoblots were also probed with actinantibodies as a loading control. C: iPLA2-specific enzymatic activity measurements inislets from WT or RIP-iPLA2-TG mice fromline TG1. Black bars represent basal specificactivity, and light gray bars represent activityin presence of ATP. Third (rightmost) bar ineach set represents activity in presence ofiPLA2 suicide substrate (E)-6-(bromo-methylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (BEL). Mean values SE (n 6)are displayed in A and C. *P 0.05 for WTvs. RIP-iPLA2-TG.




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possibility, insulin secretion by pancreatic islets isolated fromWT or RIP-iPLA2-TG male mice was compared.

Insulin secretion by pancreatic islets isolated from RIP-iPLA2-TG and iPLA2-null male mice and their respectiveWT littermates. Compared with insulin secretion at 3 mMglucose, pancreatic islets isolated from either WT or RIP-iPLA2-TG male mice were stimulated by 8 and 20 mMglucose to secrete greater amounts of insulin in a concentra-tion-dependent manner, and insulin-secretory responses wereamplified by the adenylyl cyclase activator forskolin (Fig. 8),

as expected (9, 50, 61). In the presence of forskolin, insulinsecretion stimulated by both 8 and 20 mM glucose was signif-icantly greater with islets from RIP-iPLA2-TG male micecompared with those from WT mice (Fig. 8). In contrast, isletsisolated from iPLA2-null male mice secreted less insulin inthe presence of 20 mM glucose and 2.5 M forskolin than didislets from WT mice (Fig. 9), indicating that the amplification

of insulin secretion under those conditions correlates withiPLA2 expression level.

GTT and insulin-tolerance testing of iPLA2-null male mice.

Although fasting blood glucose levels were not significantlydifferent between the two groups, blood glucose concentrationsat 60 and 120 min after intraperitoneal administration ofglucose were significantly higher in iPLA2-null than in WTmale mice (Fig. 10A), indicating that the former are lessglucose tolerant. In the fed state, blood glucose levels weresignificantly higher in iPLA2-null male mice (134 2.9mg/dl) compared with wild-type male mice (118 4.5 mg/dl)and remained significantly higher at 30 min (80 5.0 vs. 55 3.9 mg/dl) and 60 min (75 6.4 vs. 51 4.1 mg/dl) andsomewhat higher at 120 min (97 7.2 vs. 73 11.6 mg/dl)after intraperitoneal insulin administration. The fractional fallin blood glucose level was also significantly lower in iPLA2-null male mice at 30 and 60 min (to 59 3 and 56 5%,respectively, of the baseline value) than it was in WT malemice (to 47 3 and 38 5%, respectively, of the baselinevalue), reflecting impaired insulin sensitivity (Fig. 10B).

Coupled with defective amplification of insulin secretion(Fig. 9), this could account for the impaired glucose toleranceof iPLA2-null male mice (Fig. 10A), which is not observed in

female iPLA2-null mice in the absence of metabolic stressors(9). [Differences in absolute blood glucose concentrationsbetween Figs. 7 and 10 and in insulin levels between Figs. 8and 9 reflect different genetic backgrounds of RIP-iPLA2-TG(pure C57BL6/J) and iPLA2-null (mixed 129/SvJ C57BL6)mice (8) and their respective WT littermates].

Arachidonic acid incorporation and phospholipid composi-

tion of islets from RIP-iPLA2-TG male mice and their corre-sponding WT male littermates. Pancreatic islets contain anunusually high fraction of arachidonic acid-containing phos-pholipids (56), and arachidonic acid released from them isbelieved to promote insulin secretion (29, 31, 48, 5355, 74,75). It has been proposed that iPLA2 plays a critical role in

Fig. 3. Morphology and expression of iPLA2 by pancre-atic islets of RIP-iPLA2-TG or WT mice as assessed byimmunofluorescence microscopy. Sections were preparedfrom pancreata from WT (A and C) or RIP-iPLA2-TG (Band D) mice. In A and B, sections were probed withantibodies to insulin (green) and glucagon (red) and fluo-rescent secondary antibodies. In C and D, sections wereprobed with antibodies to iPLA2 (green) and glucagon(red) and fluorescent secondary antibodies.

Fig. 4. Growth of RIP-iPLA2-TG and WT male mice. Male RIP-iPLA2-TG( ) and WT () mice of ages 348 wk with free access to water and chow wereweighed between 9:00 and 10:00 AM on a top-loading balance. Mean valuesare displayed, and SD are indicated (n 4471). None of displayed valuesdiffered significantly between genotypes.




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synthesizing arachidonate-containing phospholipids by provid-ing LPC acceptors for arachidonic acid incorporation intodiradyl-GPC lipids (4, 64). It was thus considered possible thatthe enhanced insulin-secretory responses of islets from RIP-iPLA2-TG male mice (Fig. 8) might be attributable to in-creased LPC levels, accelerated incorporation of arachidonicacid, and a higher content of arachidonate-containing GPClipids to provide substrate for phospholipases activated byinsulin secretagogues, and we tested these possibilities.

Incorporation of [3H]arachidonic acid into phospholipidsoccurs at indistinguishable rates in islets from RIP-iPLA2-TGand WT male mice (Fig. 11). ESI with tandem MS analyses(ESI/MS/MS) indicated that the abundance of the major ara-

chidonate-containing GPC lipids, 16:0/20:4-GPC (m/z 788)and 18:0/20:4-GPC (m/z 816) relative to the internal standard(m/z 684), is also virtually identical in islets from RIP-iPLA2-TG and WT male mice (Fig. 12), as is their overall

content of LPC species (Fig. 13). Although there are minordifferences in relative abundances of 14:0-LPC (m/z 472) and16:0-LPC (m/z 502) relative to the internal standard (m/z 544),the relative abundances of 18:1-LPC (m/z 528) and 18:0-LPC(m/z 530) are virtually identical in RIP-iPLA2-TG and WTmouse islets (Fig. 13). These findings indicate that overexpres-sion of iPLA2 in islets does not result in any obvious pertur-bation of their arachidonate incorporation rates or phospholipidcomposition, and this is unlikely to explain their altered secre-tory responses.

Kv2.1 currents, membrane potential, and cytosolic [Ca2]of RIP-iPLA2-TG and WT male mouse islet-cells. Anothermechanism that was considered as a possible explanation for

the enhanced insulin-secretory responses of islets from RIP-iPLA2-TG mice is attenuation of Kv2.1 currents that ordi-narily serve to repolarize the -cell after secretagogue-induceddepolarization. Such repolarization limits the duration of the

Fig. 5. Fasting blood glucose and plasma insulinconcentrations in RIP-iPLA2-TG and WT malemice. Male mice 2024 wk of age were fasted over-night, and blood was obtained from lateral saphenousvein. Data are expressed as means SE (n 30).*P 0.01 for WT vs. TG mice.

Fig. 6. Oral glucose-tolerance tests with RIP-iPLA2-TGand WT male mice. A solution containing 2 mg of D-glucose per grambodyweight wasadministeredenterally to

WT and RIP-iPLA2-TG male mice 2024 wk of age bygavage. A: blood glucose concentrations at baseline and at15, 30, 60, and 120 min after glucose administration to WT() or TG ( ) mice. B: plasma insulin concentrations atbaseline and 15 min after glucose administration in WT(blackbars) and TG (gray bars) mice. Data are expressed asmeans SE (n 19). *P 0.01 for WT vs. RIP-iPLA2-TG mice.




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action potential and the associated rise in cytosolic [Ca2] thattriggers insulin secretion (28, 29). Insulinoma cells that over-express iPLA2 exhibit reduced amplitude and acceleratedinactivation of Kv2.1 channels, and these effects are mimickedby application of the PLA2 reaction product arachidonic acid(29). Mice with a disrupted Kv2.1 gene exhibit a phenotypesimilar to that of RIP-iPLA2-TG mice with mild hypoglyce-mia and hyperinsulinemia, improved glucose tolerance afterintraperitoneal glucose administration, and enhanced insulin-secretory responses of isolated islets (28). This is associated

with greatly reduced islet Kv currents, action potentials ofprolonged duration and decreased frequency, and more sus-tained periods of elevated cytosolic [Ca2] (28). We thereforeinvestigated the possibility that islets from RIP-iPLA2-TGmice would exhibit similar electrophysiological properties.

Figure 14 illustrates Kv currents for WT control (A) andRIP-iPLA2-TG islet -cells (B) elicited by 500-mS voltagesteps at 10-mV increments from 80 to 30 mV. The RIP-iPLA2-TG -cells exhibit significantly increased inactivationcompared with control -cells 10 min after we changed themedium glucose concentration from 0 to 20 mM (Fig. 14C),and the amplitude of total Kv current is also reduced in theRIP-iPLA2-TG -cells compared with control -cells.

Figure 15 illustrates islet action potential frequency duringstimulation with 20 mM glucose for WT control (A) and

RIP-iPLA2-TG islet -cells (B). The action-potential fre-quency was reduced from 1.7/s for control cells to 0.96/s ( P 0.035) for RIP-iPLA2-TG cells 5 min after glucose stimula-tion. This was associated with an increased duration of eachaction potential for the RIP-iPLA2-TG islet -cells comparedwith control cells and with a more sustained elevation in

Fig. 7. Intraperitoneal glucose and insulin tol-erance tests with RIP-iPLA2-TG and WTmale mice. In A, D-glucose (2 mg/g) and in Bhuman regular insulin (0.75 U/kg) was admin-istered by intraperitoneal injection to WT ()or RIP-iPLA2-TG ( ) male mice 2024 wkof age, and blood was collected at baseline andat 30, 60, and 120 min after injection to mea-sure glucose concentration. Values are dis-played as means SE (n 20). *P 0.05 forWT vs. TG.

Fig. 8. Insulin secretion stimulated by D-glucose and forskolin from pancreaticislets isolated from RIP-iPLA2-TG and WT mice. Islets isolated from WT(black bars) or RIP-iPLA2-TG (gray bars) male mice were incubated (30 min,37C) in buffer containing 3, 8, or 28 mM D-glucose without or with 2.5 Mforskolin, and an aliquot of medium was removed for measurement of insulin.Mean values are displayed (n 5 experiments in triplicate). *P 0.05 for WTvs. TG.

Fig. 9. Insulin secretion stimulated by D-glucose and forskolin from pancreaticislets isolated from iPLA2-null and WT mice. Islets isolated from WT (blackbars) or iPLA2-null (gray bars) male mice were incubated (30 min, 37C) inbuffer containing 3, 8, or 28 mM D-glucose without or with 2.5 M forskolin,and an aliquot of medium was removed for measurement of insulin. Meanvalues are displayed (n 5 experiments in triplicate). *P 0.05 for WT vs.iPLA2-null mice.




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cytosolic [Ca2] for the TG cells, recorded at a frequency of 10kHz from the same islet loaded with the Ca2 indicator fluo 4.The findings with Kv currents (Fig. 14) and with membranepotential and cytosolic [Ca2] (Fig. 15) with the RIP-iPLA2-TG islets are quite similar to those observed with isletsfrom Kv2.1-null mice (28), consistent with the phenotype ofimproved glucose tolerance exhibited by both of these genet-ically modified mouse lines.

DISCUSSION

These studies represent the first report of genetically modi-fied mice that overexpress iPLA2 in pancreatic islet -cells.Like other PLA2 enzymes, iPLA2 catalyzes hydrolysis of thesn-2 fatty acid substituent from glycerophospholipid substrates

(36, 64) to yield a free fatty acid (e.g., arachidonic acid) and a2-lysophospholipid (e.g., LPC) that have intrinsic mediatorfunctions (11, 51) and can initiate synthesis of other mediators

(43). Other mammalian PLA2s include the platelet-activatingfactor acetylhydrolases; the low-molecular-weight secretoryPLA2 (sPLA2) enzymes that require micromolar [Ca

2] forcatalysis (64); and group IV cytosolic PLA2 (cPLA2) enzymes(22, 47, 64, 71), of which the prototype cPLA2 preferssubstrates with sn-2 arachidonoyl residues, undergoes Ca2-induced membrane association, and is regulated by phosphor-ylation (22).

The iPLA2 is also designated group VIA PLA2 (3, 34, 67),and it does not require Ca2 for catalysis, resides in thecytoplasm of resting cells, and undergoes subcellular redistri-bution on cellular stimulation (38). It belongs to a larger classof serine lipases encoded by several genes, and all have aGXSXG lipase consensus sequence (30, 73). Various functions

have been proposed for iPLA2, including roles in phospho-lipid remodeling (2, 4) and in amplifying insulin secretion frompancreatic islet -cells (5, 36, 48, 53, 63, 66, 68). The latterhypothesis was first developed with pharmacological inhibitorsand insulinoma cell lines, and its relevance to animal physiol-ogy has now been evaluated here with genetically modifiedmice.

We have generated TG mice that overexpress iPLA2 inpancreatic islet -cells by severalfold compared with WTlittermates, and, in the fasted state, the male RIP-iPLA2-TGmice exhibit lower blood glucose and higher plasma insulinlevels than WT male mice. Upon oral or intraperitoneal glu-cose administration, the male RIP-iPLA2-TG mice develop

lower blood glucose levels than do WT male mice, althoughthe hypoglycemic responses of RIP-iPLA2-TG and WT miceto exogenous insulin are indistinguishable, suggesting thatinsulin sensitivity is not altered in RIP-iPLA2-TG mice. Isletsfrom the male RIP-iPLA2-TG mice exhibit greater amplifi-cation of glucose-induced insulin secretion by the adenylylcyclase activator forskolin than do WT islets, and this resem-bles the effect of overexpressing iPLA2 in INS-1 insulinomacells, in which the amplifying effects of agents that elevatecAMP on glucose-induced insulin secretion are substantiallygreater than in parental INS-1 cells (35). Our findings withgenetically modified mice thus support the hypothesis thatiPLA2 plays a role in amplifying insulin secretion (5, 36, 53)

Fig. 10. Intraperitoneal glucose and insulin tolerancetests with iPLA2-null and wild-type mice. In AD-glucose (2 mg/g) and in B human regular insulin(0.75 U/kg) was administered by intraperitoneal in-jection to WT () or iPLA2-null ( ) male mice2024 wk of age, and blood was collected at baselineand at 30, 60, and 120 min after injection to measureglucose concentration. Values are displayed as means SEM (n 20). *P 0.05 for WT vs. iPLA2-null mice.

Fig. 11. [3H8]arachidonic acid incorporation into glycerophosphocholine(GPC) lipids from pancreatic islets of WT and RIP-iPLA2-TG mice. WT andRIP-iPLA2-TG mouse islets were incubated with [3H8]arachidonic acid([3H]AA) for various intervals and were washed with BSA-containing bufferto remove unincorporated label. Phospholipids were extracted after labeling,and their phosphorus content was measured. GPC lipids were then isolated byTLC, and their [3H]AA content was determined by liquid scintillation spec-trometry. Values are expressed as disintegrations per minute per nanomolelipid phosphorus. Mean values SE are displayed (n 4).




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and indicate that this has demonstrable effects on in vivoglucose homeostasis in living animals.

It has been reported that introduction of the RIP-Cre con-struct, in which transcription of the sequence encoding the crerecombinase is driven by RIP, into TG mice is sufficient toinduce glucose intolerance and impaired insulin secretion evenin the absence of genes encoded by loxP sites that are the usual

targets of attempts to produce -cell-specific ablation of certaingenes (33). This raises the possibility that artifactual impair-ment of glucose homeostasis can occur as a result of geneticmanipulation that is not related to the targeted genes. In ourRIP-iPLA2-TG mice, however, improved, rather than im-paired, glucose tolerance and insulin secretion were observed,and the transgene construct did not include coding sequence forthe cre recombinase. Moreover, elimination of iPLA2 expres-sion in iPLA2-null mice produced effects in a directionopposite to those produced by overexpression of iPLA2, andthis provides independent but complementary support for thehypothesis that iPLA2 acts to augment insulin secretion andglucose tolerance.

The conclusions from experiments with INS-1 insulinoma

cells in which iPLA2 expression is suppressed by stableexpression of siRNA are also symmetrical with those involvingiPLA2-null mice. Such cell lines exhibit reduced amplifica-tion of glucose-induced insulin secretion by cAMP-elevatingagents compared with parental INS-1 cells (5). At the wholeanimal level, we find here that male iPLA2-null mice gener-ated by homologous recombination exhibit abnormal glucosetolerance in the absence of metabolic stress. Although un-stressed female iPLA2-null mice have normal glucose toler-ance, imposing the metabolic stress of high dietary fat feedingcauses greater deterioration in glucose tolerance in femaleiPLA2-null mice than in WT littermates (9).

Pancreatic islets isolated from female iPLA2-null micesubjected to high dietary fat feeding also have markedlyreduced ability to amplify glucose-induced insulin secretion inthe presence of agents that elevate cAMP (9). Here, we findthat islets from male iPLA2-null mice fed a normal-chow dietalso have reduced insulin-secretory responses to 20 mM glu-cose and the adenylyl cyclase activator forskolin, which pro-

vides further support for a role for iPLA2 in amplification ofglucose-induced insulin secretion by cAMP-elevating agents.

The opposing but complementary effects of suppressing (5,9) or increasing (35) iPLA2 expression level, respectively,with both genetically manipulated cell lines and whole animalsalso argue against the possibility that the improvements ininsulin secretion and glucose tolerance in RIP-iPLA2-TGmice reported here are nonspecific effects of iPLA2 overex-pression, although such effects can occur in overexpressionmodels.

The molecular basis for synergy between iPLA2 expressionand cAMP elevation is not yet established, but agents thatincrease -cell cAMP levels induce iPLA2 subcellular redis-tribution and association with perinuclear membranes that

include endoplasmic reticulum and Golgi (6, 36, 38). Mem-brane association could facilitate access of iPLA2 to itssubstrates, and subcellular redistribution of iPLA2 is sup-pressed by PKA inhibitors (38). Although purified, recombi-nant iPLA2 is a substrate for the catalytic subunit of PKAin vitro (38), to our knowledge PKA-catalyzed phosphoryla-tion of iPLA2 has not yet been demonstrated in intact -cellsor other cells.

The synergy between iPLA2 expression and cAMP eleva-tion in the amplification of insulin secretion from isolated isletscaused us to examine glucose tolerance after both oral andintraperitoneal administration in RIP-iPLA2-TG and WT

Fig. 12. Electrospray ionization tandem massspectrometric analyses (ESI/MS/MS) of dira-dyl-GPC lipids from pancreatic islets of WTand RIP-iPLA2-TG mice. Phospholipidsfrom pancreatic islets of WT (A) and RIP-iPLA2-TG (B) mice were mixed with inter-nal standard 14:0/14:0-GPC and were ana-lyzed as Li adducts by positive ion ESI/MS/MS scanning for constant neutral loss of183 (phosphocholine), and relative abun-dances of ion currents were plotted vs. m/zvalue.

Fig. 13. ESI/MS/MS of the lysophosphatidyl-choline (LPC) content of pancreatic islets ofWT and RIP-iPLA2-TG mice. Phospholipidswere extracted from pancreatic islets of WT(A) and RIP-iPLA2-TG (B) mice, mixed withinternal standard 19:0-LPC, and analyzed asLi adducts by positive ion ESI/MS/MS scan-ning for constant neutral loss of 59 (trimeth-ylamine) to visualize LPC species.




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mice. We reasoned that the incretin effect of gut peptidesreleased after oral glucose administration might increase isletcAMP and cause greater amplification of insulin secretion from

RIP-iPLA2-TG islets than that which occurred after intraperi-toneal glucose administration. Although the glucose tolerance

of RIP-iPLA2-TG mice was greater than that of WT miceboth in oral and intraperitoneal GTT, the difference betweenthe genotypes was not greater in the oral test. Among the

possibilities these results could reflect is that the incretin effectis not as strong as expected, that countervailing adrenergic

Fig. 14. Delayed rectifier currents in pancreatic islet-cells from WT control and RIP-iPLA2-TG mice.Kv current traces recorded from islet -cells from WTcontrol (A) or RIP-iPLA2-TG mice (B) incubated inmedium without glucose (left tracings) or with 20 mMglucose (right tracings) and subjected to 500-ms de-polarization in 10 mV and 5 mV increments from80mV to 30 mV are shown. In C, fold increase in Kvinactivation percentage is expressed as a function ofdepolarizing voltage for WT control (black bars) orRIP-iPLA2-TG islet -cells (gray bars) at 10 minafter switching medium glucose concentration from 0to 20 mM. Mean values are displayed, and error barsrepresent SD (n 7).

Fig. 15. RIP-iPLA2-TG mouse islets have increased glucose-induced action potential duration with decreased frequency and corresponding changes inglucose-induced elevation of cytosolic Ca2 concentration ([Ca2]) compared with WT control islets. Electrical activity in response to 20 mM glucose is recordedfor WT control (A) or RIP-iPLA2-TG (B) mouse islet attached -cells. Insets display action potentials (APs) from segment of activity indicated by horizontalbars. Lowest set of tracings in each panel represents fast-acquisition [Ca2] traces recorded from same entire mouse islet during segment of activity indicatedby horizontal bars, loaded with fluo 4, and imaged at a frequency of 10 kHz. Decrease in AP frequency of WT control vs. RIP-iPLA2-TG mouse islets (from1.7/s to 0.96/s) taken at 5 min after glucose was significant (P 0.035, n 6 each).




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influences supervene because of the stress of oral gavage,because of overriding autonomic effects, or for other reasonsoperative in vivo that do not affect the behavior of isolatedislets ex vivo.

Several events that occur as a consequence of iPLA2-catalyzed phospholipid hydrolysis and accumulation of nones-terified arachidonate and LPC could contribute to amplification

of secretagogue-induced rises in -cell [Ca2

] and insulinsecretion. These include facilitating Ca2 entry (53, 54, 75) byarachidonate effects on voltage-operated Ca2 channels (72),effects of LPC and arachidonate on KATP (18), effects of anarachidonate 12-lipoxygenase product on a plasma membraneNa-K-ATPase (48), and either direct effects of LPC (65) orindirect effects of arachidonate (74) on plasma membranestore-operated cation channels (17).

A recently demonstrated molecular mechanism throughwhich iPLA2 contributes to regulation of -cell membranepotential and Ca2 transients is that arachidonate released byiPLA2 inactivates voltage-operated Kv2.1 channels in the-cell plasma membrane and thereby increases the duration ofdepolarization and the associated rise in [Ca2] induced bysecretagogues (29). This demonstration rests in part on findingswith INS-1 cells, in which exogenous arachidonate suppressesKv2.1 currents (29). In addition, INS-1 cells that overexpressiPLA2 exhibit reduced depolarization-induced Kv2.1 cur-rents, and such currents are enhanced in iPLA2-null -cells(29). Such effects of iPLA2 prolong secretagogue-induceddepolarization and Ca2 entry into -cells and amplify theinsulin-secretory response.

Here we demonstrate that islets from RIP-iPLA2-TG micealso exhibit reduced and more rapidly inactivating Kv2.1currents than do WT control mouse islets. This is associatedwith reduced frequency and increased duration of glucose-induced action potentials in the TG islet -cells, and there is a

corresponding prolongation of the glucose-induced rise incytosolic [Ca2]. These findings are quite similar to recentlyreported electrophysiological properties of islets from Kv2.1-null mice (28). Those mice also exhibit a phenotype similar toRIP-iPLA2-TG mice with respect to glucose homeostasis,including mild fasting hypoglycemia and hyperinsulinemia,improved glucose tolerance after intraperitoneal glucose ad-ministration, and enhanced insulin-secretory responses of iso-lated islets (28). These observations suggest that iPLA2contributes to physiologically significant regulation of -cellmembrane potential and Ca2 transients in part by restrainingKv2.1 delayed rectifier channel activity and that this has ademonstrable impact on glucose homeostasis at the wholeanimal level.

Pancreatic islet -cells express PLA2 enzymes in addition toiPLA2, including the group IB secretory PLA2 (sPLA2-IB),which resides in part in insulin-secretory granules and iscosecreted with insulin (57). Although iPLA2 and sPLA2-IBboth hydrolyze the sn-2 fatty acid substituents from glycero-lipid substrates to yield a free fatty acid and a 2-lysophospho-lipid, the two enzymes reside in different compartments andhave different catalytic requirements. iPLA2 is active atnanomolar to low micromolar Ca2 concentrations that occurwithin the cytosol of living cells, resides predominantly incytosol in resting cells, and undergoes subcellular redistribu-tion to associate with membranous organelles on stimulation of-cells with secretagogues (35, 38). In contrast, sPLA2-IB

requires millimolar concentrations of Ca2 for catalytic activ-ity and functions as an extracellular secreted enzyme (64).

sPLA2-IB-null mice have been prepared and found to beresistant to high-fat-diet-induced obesity and insulin resistanceand to exhibit reduced postprandial hyperglycemia, superiorglucose tolerance, and increased insulin sensitivity comparedwith WT littermates (27, 32). These effects appear to be

attributable to reduced absorption and blood levels of lyso-phospholipids, such as LPC, and to improved glucose uptakeby several peripheral tissues, including liver, heart, and skeletalmuscle (32). Plasma insulin levels after intraperitoneal glucoseadministration are slightly but not significantly lower insPLA2-IB-null mice than in WT mice, and the improvedglucose tolerance of the sPLA2-IB-null mice is thought toreflect principally improved insulin sensitivity (32).

A widely cited hypothesis is that iPLA2 is critical forsynthesis of arachidonate-containing phospholipids and pro-vides LPC acceptors for incorporating arachidonic acid intodiradyl-GPC lipids (4, 64). This could be important becausepancreatic islets have the highest arachidonate-containingphospholipid content of any known tissue (56), and arachi-donic acid released by secretagogue-activated phospholipasesis thought to promote insulin secretion (29, 31, 48, 53, 54, 74,75). If iPLA2 overexpression increased the content of arachi-donate-containing phospholipids, this might contribute to en-hanced RIP-iPLA2-TG islet secretion, but [

3H]arachidonateincorporation and content of LPC and arachidonate-containingGPC lipids of WT and RIP-iPLA2-TG islets are indistin-guishable. This is consistent with data from INS-1 cells thatoverexpress (35) or underexpress (5) iPLA2 and from severaltissues and cells from iPLA2-null mice, including islets (9),testes (8), and peritoneal macrophages (7). These findings callinto question the relevance of the iPLA2-arachidonate incor-poration hypothesis to the physiology of normal cells because

it is based on findings with transformed, cultured, macrophage-like cells (4, 64), but the process does not operate in nativemacrophages (7).

In conclusion, the findings reported here with TG mice thatoverexpress iPLA2 in pancreatic islet -cells support thehypothesis that iPLA2 plays a role in amplifying insulinsecretion and indicate that this is relevant to the physiology ofglucose homeostasis in living animals.

ACKNOWLEDGMENTS

We thank Sheng Zhang and Min Tan for excellent technical assistance, Dr.Sasanka Ramanadham for a critical reading of the manuscript, and Dr. RichardGross for iPLA2 antibody 506. We also gratefully acknowledge O. Pongs,S. A. Goldstein, L. E. Fridlyand, J. P. Lopez, and N. A. Tamarina for helpfuldiscussions and suggestions and F. Mendez and S. Eames for technical

expertise and assistance.

GRANTS

This work was supported by United States Public Health Service GrantsR37-DK-34388, P41-RR-00954, P60-DK-20579, and P30-DK-56341 to J.Turk; by DK-48494 to L. H. Philipson; and by University of Chicago grantDRTC DK-20595. D. A. Jacobson was supported in part by a postdoctoralfellowship in -cell research from Takeda Pharmaceuticals North America.

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Glucose homeostasis, insulin secretion, and islet phospholipids in mice

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