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EDITORIAL FOCUS
1Division of Endocrinology, Metabolism, and Lipid Research, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri; and 2Department of Medicine, University of Chicago, Chicago, Illinois
Submitted 23 July 2007 ; accepted in final form 23 September 2007
ABSTRACT
Studies with genetically modified insulinoma cells suggest that group VIA phospholipase A2 (iPLA2β) participates in amplifying glucose-induced insulin secretion. INS-1 insulinoma cells that overexpress iPLA2β, for example, exhibit amplified insulin-secretory responses to glucose and cAMP-elevating agents. To determine whether similar effects occur in whole animals, we prepared transgenic (TG) mice in which the rat insulin 1 promoter (RIP) drives iPLA2β overexpression, and two characterized TG mouse lines exhibit similar phenotypes. Their pancreatic islet iPLA2β expression is increased severalfold, as reflected by quantitative PCR of iPLA2β mRNA, immunoblotting of iPLA2β protein, and iPLA2β enzymatic activity. Immunofluorescence microscopic studies of pancreatic sections confirm iPLA2β overexpression in RIP-iPLA2β-TG islet β-cells without obviously perturbed islet morphology. Male RIP-iPLA2β-TG mice exhibit lower blood glucose and higher plasma insulin concentrations than wild-type (WT) mice when fasting and develop lower blood glucose levels in glucose tolerance tests, but WT and TG blood glucose levels do not differ in insulin tolerance tests. Islets from male RIP-iPLA2β-TG mice exhibit greater amplification of glucose-induced insulin secretion by a cAMP-elevating agent than WT islets. In contrast, islets from male iPLA2β-null mice exhibit blunted insulin secretion, and those mice have impaired glucose tolerance. Arachidonate incorporation into and the phospholipid composition of RIP-iPLA2β-TG islets are normal, but they exhibit reduced Kv2.1 delayed rectifier current and prolonged glucose-induced action potentials and elevations of cytosolic Ca2+ concentration that suggest a molecular mechanism for the physiological role of iPLA2β to amplify insulin secretion.
transgenic mice; glucose tolerance; insulin tolerance
Understanding control of β-cell growth, death, and secretion might permit iterative introduction of genes into β-cell lines or precursors to optimize secretion, proliferation, and resistance to injury (24, 69). Combined with empirical selection and encapsulation, such β-cell engineering might someday provide a renewable source of β-cells for replacement therapy (44, 45). Developing such potential future therapies requires increased understanding of the genes and gene products that govern β-cell physiology (46).
Insulin secretion involves glucose transport into β-cells, phosphorylation by glucokinase at a rate proportional to extracellular glucose concentrations, and further metabolism that increases the ATP/ADP concentration ratio (20, 39, 40). This inactivates β-cell ATP-sensitive K channels (KATP), causing membrane depolarization, voltage-operated Ca2+-channel opening, and a rise in Ca2+ concentration ([Ca2+]) that induces exocytosis (1, 14, 21). Several other, incompletely understood processes modulate or amplify insulin secretion, e.g., nonselective cation channels activated by Ca2+-store depletion, an electrogenic Na-K-ATPase, and voltage-operated K channels also affect β-cell membrane potential and secretion (17, 23, 29, 48).
Reducing equivalent shuttles to and from mitochondria are also required for secretion (19, 58, 76), and glucose augments secretion by KATP-independent mechanisms (60, 77). Elevating β-cell cAMP also amplifies secretion by protein kinase A (PKA)-dependent and -independent mechanisms (50, 61), and β-cell signaling involves phospholipid hydrolysis and accumulation of phospholipid-derived mediators (31, 41, 48, 50, 52, 63, 68, 70).
We and others (48, 68, 70) have developed evidence that an islet phospholipase A2 that does not require Ca2+ (iPLA2β) is activated by secretagogues and that its products participate in β-cell signaling. Characterizing this activity resulted in our cloning an 84-kDa phospholipase A2 from islet cDNA libraries (34, 37). Recombinant iPLA2β is Ca2+ independent, activated by ATP, and inhibited by a suicide substrate that also attenuates glucose-induced insulin secretion, arachidonate release, and rises in β-cell [Ca2+] (52).
Genetic gain- and loss-of-function studies with insulinoma cells support the possible involvement of iPLA2β in insulin secretion. INS-1 insulinoma cells stably transfected to express short interfering RNA (siRNA) that reduces iPLA2β expression exhibit reduced insulin secretion when stimulated with glucose and markedly impaired amplification of insulin secretion by cAMP-elevating agents (5). In contrast, stably transfected INS-1 cells that overexpress iPLA2β exhibit amplified insulin-secretory responses to glucose, particularly in the presence of cAMP-elevating agents, and this is associated with subcellular redistribution and membrane association of iPLA2β (35).
To determine whether these findings with cultured insulinoma cells are relevant to animal physiology, we have prepared transgenic (TG) mice in which iPLA2β overexpression is driven by the rat insulin 1 promoter (RIP) and studied fasting concentrations of glucose and insulin, blood glucose excursions on administration of glucose or exogenous insulin, and insulin-secretory and electrophysiological responses of pancreatic islets isolated from RIP-iPLA2β-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 (Ann Arbor, MI); enhanced chemiluminescence reagents from Amersham Biosciences (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' balanced salt solution, L-glutamine, agarose, molecular mass standards, and RT-PCR reagents from Invitrogen (Carlsbad, CA); fetal bovine serum from Hyclone (Logan, UT); Pentex bovine serum albumin (BSA, fatty acid-free, fraction V) from ICN Biomedical (Aurora, OH); and forskolin from Calbiochem (La Jolla, CA). Krebs-Ringer bicarbonate (KRB) buffer contained (in mM) 25 HEPES (pH 7.4), 115 NaCl, 24 NaHCO3, 5 KCl, 1 MgCl2, and 2.5 CaCl2.
Generation and genotyping of RIP-iPLA2β-TG mice. As described previously (10), full-length iPLA2β cDNA was inserted in a RIP-I/β-globin expression vector and was microinjected into fertilized eggs of C57BL/6J mice. TG founders were mated with WT C57BL/6J mice (Jackson Laboratory), and N2 and N3 generations from two lines with similar phenotypes were studied. The genetic background of resultant mice was pure C57BL/6J, and WT littermates were used as controls. All protocols were approved by the Washington University Animal Studies Committee.
Genotyping was performed with tail-clipping DNA either by Southern blotting analyses or by PCR. For Southern blots, DNA was digested with EcoR I restriction endonuclease. Digests were analyzed by electrophoresis and were transferred to nylon membranes, which were incubated with a 32P-labeled probe that recognizes the sequence in the rabbit hemoglobin gene contained in the original construct. For PCR 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 CAG and (Fabpi 3') TAG AGC TTT GCC ACA TCA CAG GTC ATT CAG. The expected size of the product was 450 bp. The other primer pair amplified the sequence that spans the junction of iPLA2β and globin 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 gta ttt gtg agc cag gg. The expected size of the product was 200 bp.
Generating and genotyping iPLA2β-null mice. Preparation of the iPLA2β knockout construct, its introduction into 129/SvJ mouse embryonic stem cells, their selection with G418, characterization by Southern blotting, injection into C57BL/6 mouse blastocysts, the production of chimeras and then heterozygotes, and the mating of heterozygotes to yield WT, heterozygous, and iPLA2β-null mice in a Mendelian distribution are described elsewhere, as is their genotyping by Southern blotting of tail genomic DNA (7–9). The genetic background of the resultant mice is mixed 129/SvJ x C57BL/6.
Islet isolation. Islets were isolated from pancreata of male WT, RIP-iPLA2β-TG, and iPLA2β-null mice by collagenase digestion after mincing, followed by Ficoll step density gradient separation and manual selection under stereomicroscopic visualization to exclude contaminating tissues (9, 49). Mouse islets were counted and used for PCR and immunoblotting of iPLA2β mRNA and protein, respectively; for measuring iPLA2β-specific enzymatic activity and ex vivo secretion of insulin and electrophysiological responses; and for extraction of phospholipids.
PCR of iPLA2β mRNA in mouse islets. As described previously (9, 13), total RNA was extracted with TRIzol reagent (Invitrogen). After treatment with DNase I, 1 µg of total RNA was reverse transcribed with an oligo(dT) primer, and quantitative PCR was performed with an ABI Prism 7700 PCR instrument (Applied Biosystems) using the SYBR 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 a fragment of iPLA2β cDNA. The sequence of primer 1 was GCC CTG GCC ATT CTA CAC AGT A, and that of primer 2 was CAC CTC ATC CTT CAT ACG GAA GT. Amplification specificity was verified by agarose gel electrophoresis of products and a heat-dissociation protocol. Sequence-specific amplification was detected with increasing fluorescent signal of FAM (reporter dye) during the amplification cycle. The amplification of 18s rRNA served as control.
Western-blotting analyses. As described previously (35), proteins in islet homogenates were analyzed by SDS-PAGE (7.5%), transferred onto a PVDF membrane, and probed with antibody 506 provided by Dr. Richard Gross (Washington University, St. Louis, MO). Protein bands were visualized by ECL. Membranes were also probed 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 by Bio-Rad assay, and iPLA2 activity was measured in aliquots (
20 µg protein) added to assay buffer (200 mM Tris·HCl, pH 7.0; total assay volume, 200 µl) containing 5 mM EGTA with or without 1 mM ATP. Some aliquots were pretreated (2 min) with BEL (10 µM) before assay. Reactions were initiated by injecting substrate (L-
-1-palmitoyl-2-[14C]arachidonoyl-phosphatidylethanolamine; specific activity, 50 Ci/mol; final concentration, 5 µM) in ethanol (5 µl). Assay mixtures were incubated (3 min, 37°C), and reactions were terminated by adding butanol (0.1 ml) and vortexing. After centrifugation (2,000 g, 4 min), products in the butanol layer were analyzed by silica gel G TLC in hexane/ethyl ether/acetic acid (80:20:1). The TLC region containing free arachidonic acid (Rf, 0.58) was scraped into vials, and its 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 for 60 s; Vibra Cell probe sonicator; Sonics and Materials, Danbury, CT). After centrifugation (2,800 g, 5 min) to remove tissue debris, supernatants were transferred to silanized 10-ml glass tubes and were extracted with methanol (1 ml), chloroform (1 ml), and water (1.8 ml). Samples were vortex mixed and centrifuged (900 g, 5 min). Supernatants 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 on glass 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 secondary antibody. Immunostaining for glucagon was performed with rabbit anti-glucagon (1:500; Chemicon International, Temecula, CA) as the primary antibody. Incubations with primary antibodies were performed overnight in a humidified chamber. Secondary antibodies were donkey anti-guinea pig FITC (for insulin) and donkey anti-rabbit Cy3 (for glucagon). Slides were examined with a Nikon TE300 microscope.
Fasting and fed blood glucose and insulin concentrations. As described previously (10), blood samples were obtained from the lateral saphenous vein in heparinized capillary tubes, and glucose concentrations were measured in whole blood with a blood-glucose monitor (Becton Dickenson) or an Ascensia ELITE XL blood-glucose meter. Plasma was prepared from heparinized blood by centrifugation, and insulin levels were determined in aliquots (5 µl) with a rat insulin ELISA kit (Crystal Chem). Fasting blood samples were obtained after an overnight fast, and fed blood samples were obtained between 9:00 and 10:00 AM.
Glucose and insulin tolerance tests. As described (9), intraperitoneal glucose tolerance tests (GTT) were performed in mice fasted overnight from which a baseline blood sample was obtained, followed by intraperitoneal injection of D-glucose (2 mg/g) and collection of blood for measurement of glucose after 30, 60, and 120 min. Oral GTT (42) were performed in a similar manner except that glucose (2 mg/kg) was administered enterally by gavage. Insulin tolerance tests were performed in mice with free access to water and chow that received an intraperitoneal injection (0.75 U/kg) of human regular insulin (Lilly, Indianapolis, IN), followed by collection of blood after 30, 60, and 120 min for glucose determinations (9, 49).
Insulin secretion by isolated pancreatic islets. Islets were isolated from pancreata of male mice as described in Islet isolation. Insulin-secretion studies were performed with 30 islets per incubation, as described previously (9, 49). Islets were rinsed with KRB medium containing 3 mM glucose and 0.1% BSA and were placed in silanized tubes (12 x 75 mm) in the same buffer, through which 95% air-5% CO2 was bubbled before incubations. Tubes were capped and incubated (37°C, 30 min) in a shaking water bath. Buffer was then replaced with KRB medium containing 3, 8, or 20 mM glucose and 0.1% BSA without or with forskolin (2.5 µM), and samples were incubated for 30 min. Secreted insulin was measured by radioimmunoassay.
Determination of [3H]arachidonic acid incorporation into islet phospholipids. As described earlier (55), islets were washed thrice in KRB medium containing 5.5 mM glucose and 0.1% BSA, resuspended in that medium, and preincubated for 30 min at 37°C. Islets (100 per condition) were then placed in fresh KRB medium that contained 5.5 mM glucose, 0.1% BSA, and 2.5 mM CaCl2, and [3H]arachidonic acid (final concentration 0.5 µCi/ml, 5 nM) was added to the medium. Incubations were performed for 10–60 min at 37°C, and islets were then washed thrice in KRB medium containing 5.5 mM glucose and 0.1% BSA to remove unincorporated [3H]arachidonic acid. Phospholipids were then extracted as described above and were analyzed by TLC, and the [3H]arachidonate content of glycerophosphocholine (GPC) lipids was determined by liquid scintillation spectrometry and normalized to lipid phosphorus content.
Positive ion electrospray ionization mass spectrometric analyses of choline-containing glycerolipids. Diradyl-GPC lipids and lysophosphatidylcholine (LPC) species were analyzed as Li+ adducts by positive ion electrospray ionization mass spectrometric analyses (ESI/MS) on a Finnigan (San Jose, CA) TSQ-7000 triple-stage quadrupole mass spectrometer with an ESI source controlled by Finnigan ICIS software. Phospholipids were dissolved in methanol/chloroform (2/1, vol/vol) containing LiOH (10 pmol/µl), infused (1 µl/min) with a Harvard syringe pump, and analyzed as described (25, 26). For tandem MS, precursor ions selected in the first quadrupole were accelerated (32–36 eV collision energy) into a chamber containing argon (2.3–2.5 mTorr) to induce collision-activated dissociation, and product ions were analyzed in the final quadrupole. Identities of GPC species were determined from their tandem spectra (25, 26), and their quantities were determined relative to internal standards by interpolation from standard curves (5, 55).
Whole cell ruptured-patch electrophysiological recording.
Voltage-activated currents were recorded by using whole cell ruptured-patch clamps with an Axopatch 200B amplifier and pCLAMP 9 software (Molecular Devices), as described (28, 29). Patch electrodes (2–4 M
) were loaded with intracellular solution containing (in mmol/l) 140 KCl, 1 MgCl2[H2O]6, 10 EGTA, 10 HEPES, 5 MgATP (pH 7.25) with KOH. Islets were perfused with KRB containing (in mmol/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 the indicated glucose concentration.
Islet perforated-patch electrophysiological recording.
Patch electrodes (2–4 M) were loaded with intracellular solution containing (in mmol/l) 140 KCl, 1 MgCl2[H2O]6, 10 EGTA, 10 HEPES (pH 7.25) with KOH containing the pore-forming antibiotic amphotericin B (52) (Sigma), as described (28, 29). Islets were perfused with KRB containing (in mmol/l) 119 NaCl, 2 CaCl2[(H2O)6], 4.7 KCl, 10 HEPES, 1.2 MgSO4, 1.2 KH2PO4, and glucose as indicated, adjusted to pH 7.3 with NaOH. Cells on the periphery of islets on glass coverslips were sealed in voltage clamp at –80 V, and the amphotericin allowed perforation and good access over several minutes. After being switched to current clamp, cells that had a resting membrane voltage near –65 mV in 2 mM glucose and near –75 mV in 0 mM glucose were assumed to be β-cells. The glucose-containing solution (at 37°C) was perfused into a heated chamber (at 37°C).
High-speed intracellular calcium imaging. Mouse islets attached to glass-bottom Matek tissue-culture dishes were loaded with 5 µM fluo 4-AM loaded in Krebs-Ringer-Henseleit 2 buffer for 30 min at 37°C, as described (28, 29). Loaded islets were placed in a temperature controller (TC-202; Medical Systems) mounted on the stage of an inverted microscope (Nikon Eclipse TE2000-U) for imaging. Experiments were performed at 37°C. Islets were excited at 488 nm from a monochrometer (Till Photonics), and the emitted light was filtered with a 535/40 filter (Chroma) and recorded with a photomultiplier tube (Photon Technology International). Acquisition was controlled with pCLAMP 9 software. In all experiments, to obtain data with sufficiently high time resolution, we used 10-kHz acquisition. Data analysis was performed with pCLAMP 9 and Microsoft Excel.
Statistical methods. Results are presented as means ± SE. Data were evaluated by unpaired, two-tailed Student's t-test or by analysis of variance with appropriate post hoc tests. Significance levels are described in figure legends.
RESULTS
Generation of TG mice that overexpress iPLA2β in pancreatic islet β-cells. In the construct used to generate the TG mice (Fig. 1A), rat iPLA2β cDNA was inserted downstream of the RIP at a site within the rabbit globin gene sequence. Transcription of the sequence encoding iPLA2β is under control of RIP, and TG overexpression of iPLA2β is expected in cells that express insulin, e.g., pancreatic islet β-cells, but not in other cells. Transgene incorporation was determined by Southern blotting (Fig. 1B) and PCR (Fig. 1C) analyses. Two founders were identified, and progeny from each were viable and fertile. Mice from TG lines TG1 and TG2 exhibited similar phenotypes.
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Arachidonic acid incorporation and phospholipid composition of islets from RIP-iPLA2β-TG male mice and their corresponding WT male littermates. Pancreatic islets contain an unusually high fraction of arachidonic acid-containing phospholipids (56), and arachidonic acid released from them is believed to promote insulin secretion (29, 31, 48, 53–55, 74, 75). It has been proposed that iPLA2β plays a critical role in synthesizing arachidonate-containing phospholipids by providing LPC acceptors for arachidonic acid incorporation into diradyl-GPC lipids (4, 64). It was thus considered possible that the enhanced insulin-secretory responses of islets from RIP-iPLA2β-TG male mice (Fig. 8) might be attributable to increased LPC levels, accelerated incorporation of arachidonic acid, and a higher content of arachidonate-containing GPC lipids to provide substrate for phospholipases activated by insulin secretagogues, and we tested these possibilities.
Incorporation of [3H]arachidonic acid into phospholipids occurs at indistinguishable rates in islets from RIP-iPLA2β-TG and WT male mice (Fig. 11). ESI with tandem MS analyses (ESI/MS/MS) indicated that the abundance of the major arachidonate-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-iPLA2β-TG and WT male mice (Fig. 12), as is their overall content of LPC species (Fig. 13). Although there are minor differences in relative abundances of 14:0-LPC (m/z 472) and 16:0-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-iPLA2β-TG and WT mouse islets (Fig. 13). These findings indicate that overexpression of iPLA2β in islets does not result in any obvious perturbation of their arachidonate incorporation rates or phospholipid composition, and this is unlikely to explain their altered secretory responses.
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Figure 14 illustrates Kv currents for WT control (A) and RIP-iPLA2β-TG islet β-cells (B) elicited by 500-mS voltage steps at 10-mV increments from –80 to 30 mV. The RIP-iPLA2β-TG β-cells exhibit significantly increased inactivation compared with control β-cells 10 min after we changed the medium glucose concentration from 0 to 20 mM (Fig. 14C), and the amplitude of total Kv current is also reduced in the RIP-iPLA2β-TG β-cells compared with control β-cells.
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These studies represent the first report of genetically modified mice that overexpress iPLA2β in pancreatic islet β-cells. Like other PLA2 enzymes, iPLA2β catalyzes hydrolysis of the sn-2 fatty acid substituent from glycerophospholipid substrates (36, 64) to yield a free fatty acid (e.g., arachidonic acid) and a 2-lysophospholipid (e.g., LPC) that have intrinsic mediator functions (11, 51) and can initiate synthesis of other mediators (43). Other mammalian PLA2s include the platelet-activating factor acetylhydrolases; the low-molecular-weight secretory PLA2 (sPLA2) enzymes that require micromolar [Ca2+] for catalysis (64); and group IV cytosolic PLA2 (cPLA2) enzymes (22, 47, 64, 71), of which the prototype cPLA2 prefers substrates with sn-2 arachidonoyl residues, undergoes Ca2+-induced membrane association, and is regulated by phosphorylation (22).
The iPLA2β is also designated group VIA PLA2 (3, 34, 67), and it does not require Ca2+ for catalysis, resides in the cytoplasm of resting cells, and undergoes subcellular redistribution on cellular stimulation (38). It belongs to a larger class of serine lipases encoded by several genes, and all have a GXSXG lipase consensus sequence (30, 73). Various functions have been proposed for iPLA2β, including roles in phospholipid remodeling (2, 4) and in amplifying insulin secretion from pancreatic islet β-cells (5, 36, 48, 53, 63, 66, 68). The latter hypothesis was first developed with pharmacological inhibitors and insulinoma cell lines, and its relevance to animal physiology has now been evaluated here with genetically modified mice.
We have generated TG mice that overexpress iPLA2β in pancreatic islet β-cells by severalfold compared with WT littermates, and, in the fasted state, the male RIP-iPLA2β-TG mice exhibit lower blood glucose and higher plasma insulin levels than WT male mice. Upon oral or intraperitoneal glucose administration, the male RIP-iPLA2β-TG mice develop lower blood glucose levels than do WT male mice, although the hypoglycemic responses of RIP-iPLA2β-TG and WT mice to exogenous insulin are indistinguishable, suggesting that insulin sensitivity is not altered in RIP-iPLA2β-TG mice. Islets from the male RIP-iPLA2β-TG mice exhibit greater amplification of glucose-induced insulin secretion by the adenylyl cyclase activator forskolin than do WT islets, and this resembles the effect of overexpressing iPLA2β in INS-1 insulinoma cells, in which the amplifying effects of agents that elevate cAMP on glucose-induced insulin secretion are substantially greater than in parental INS-1 cells (35). Our findings with genetically modified mice thus support the hypothesis that iPLA2β plays a role in amplifying insulin secretion (5, 36, 53) and indicate that this has demonstrable effects on in vivo glucose homeostasis in living animals.
It has been reported that introduction of the RIP-Cre construct, in which transcription of the sequence encoding the cre recombinase is driven by RIP, into TG mice is sufficient to induce glucose intolerance and impaired insulin secretion even in the absence of genes encoded by loxP sites that are the usual targets of attempts to produce β-cell-specific ablation of certain genes (33). This raises the possibility that artifactual impairment of glucose homeostasis can occur as a result of genetic manipulation that is not related to the targeted genes. In our RIP-iPLA2β-TG mice, however, improved, rather than impaired, glucose tolerance and insulin secretion were observed, and the transgene construct did not include coding sequence for the cre recombinase. Moreover, elimination of iPLA2β expression in iPLA2β-null mice produced effects in a direction opposite to those produced by overexpression of iPLA2β, and this provides independent but complementary support for the hypothesis that iPLA2β acts to augment insulin secretion and glucose tolerance.
The conclusions from experiments with INS-1 insulinoma cells in which iPLA2β expression is suppressed by stable expression of siRNA are also symmetrical with those involving iPLA2β-null mice. Such cell lines exhibit reduced amplification of glucose-induced insulin secretion by cAMP-elevating agents compared with parental INS-1 cells (5). At the whole animal level, we find here that male iPLA2β-null mice generated by homologous recombination exhibit abnormal glucose tolerance in the absence of metabolic stress. Although unstressed female iPLA2β-null mice have normal glucose tolerance, imposing the metabolic stress of high dietary fat feeding causes greater deterioration in glucose tolerance in female iPLA2β-null mice than in WT littermates (9).
Pancreatic islets isolated from female iPLA2β-null mice subjected to high dietary fat feeding also have markedly reduced ability to amplify glucose-induced insulin secretion in the presence of agents that elevate cAMP (9). Here, we find that islets from male iPLA2β-null mice fed a normal-chow diet also have reduced insulin-secretory responses to 20 mM glucose and the adenylyl cyclase activator forskolin, which provides further support for a role for iPLA2β in amplification of glucose-induced insulin secretion by cAMP-elevating agents.
The opposing but complementary effects of suppressing (5, 9) or increasing (35) iPLA2β expression level, respectively, with both genetically manipulated cell lines and whole animals also argue against the possibility that the improvements in insulin secretion and glucose tolerance in RIP-iPLA2β-TG mice reported here are nonspecific effects of iPLA2β overexpression, although such effects can occur in overexpression models.
The molecular basis for synergy between iPLA2β expression and cAMP elevation is not yet established, but agents that increase β-cell cAMP levels induce iPLA2β subcellular redistribution and association with perinuclear membranes that include endoplasmic reticulum and Golgi (6, 36, 38). Membrane association could facilitate access of iPLA2β to its substrates, and subcellular redistribution of iPLA2β is suppressed by PKA inhibitors (38). Although purified, recombinant iPLA2β is a substrate for the catalytic subunit of PKA in vitro (38), to our knowledge PKA-catalyzed phosphorylation of iPLA2β has not yet been demonstrated in intact β-cells or other cells.
The synergy between iPLA2β expression and cAMP elevation in the amplification of insulin secretion from isolated islets caused us to examine glucose tolerance after both oral and intraperitoneal administration in RIP-iPLA2β-TG and WT mice. We reasoned that the incretin effect of gut peptides released after oral glucose administration might increase islet cAMP and cause greater amplification of insulin secretion from RIP-iPLA2β-TG islets than that which occurred after intraperitoneal glucose administration. Although the glucose tolerance of RIP-iPLA2β-TG mice was greater than that of WT mice both in oral and intraperitoneal GTT, the difference between the genotypes was not greater in the oral test. Among the possibilities these results could reflect is that the incretin effect is not as strong as expected, that countervailing adrenergic influences supervene because of the stress of oral gavage, because of overriding autonomic effects, or for other reasons operative in vivo that do not affect the behavior of isolated islets ex vivo.
Several events that occur as a consequence of iPLA2β-catalyzed phospholipid hydrolysis and accumulation of nonesterified arachidonate and LPC could contribute to amplification of secretagogue-induced rises in β-cell [Ca2+] and insulin secretion. These include facilitating Ca2+ entry (53, 54, 75) by arachidonate effects on voltage-operated Ca2+ channels (72), effects of LPC and arachidonate on KATP (18), effects of an arachidonate 12-lipoxygenase product on a plasma membrane Na-K-ATPase (48), and either direct effects of LPC (65) or indirect effects of arachidonate (74) on plasma membrane store-operated cation channels (17).
A recently demonstrated molecular mechanism through which iPLA2β contributes to regulation of β-cell membrane potential and Ca2+ transients is that arachidonate released by iPLA2β inactivates voltage-operated Kv2.1 channels in the β-cell plasma membrane and thereby increases the duration of depolarization and the associated rise in [Ca2+] induced by secretagogues (29). This demonstration rests in part on findings with INS-1 cells, in which exogenous arachidonate suppresses Kv2.1 currents (29). In addition, INS-1 cells that overexpress iPLA2β exhibit reduced depolarization-induced Kv2.1 currents, and such currents are enhanced in iPLA2β-null β-cells (29). Such effects of iPLA2β prolong secretagogue-induced depolarization and Ca2+ entry into β-cells and amplify the insulin-secretory response.
Here we demonstrate that islets from RIP-iPLA2β-TG mice also exhibit reduced and more rapidly inactivating Kv2.1 currents than do WT control mouse islets. This is associated with reduced frequency and increased duration of glucose-induced action potentials in the TG islet β-cells, and there is a corresponding prolongation of the glucose-induced rise in cytosolic [Ca2+]. These findings are quite similar to recently reported electrophysiological properties of islets from Kv2.1-null mice (28). Those mice also exhibit a phenotype similar to RIP-iPLA2β-TG mice with respect to glucose homeostasis, including mild fasting hypoglycemia and hyperinsulinemia, improved glucose tolerance after intraperitoneal glucose administration, and enhanced insulin-secretory responses of isolated islets (28). These observations suggest that iPLA2β contributes to physiologically significant regulation of β-cell membrane potential and Ca2+ transients in part by restraining Kv2.1 delayed rectifier channel activity and that this has a demonstrable impact on glucose homeostasis at the whole animal level.
Pancreatic islet β-cells express PLA2 enzymes in addition to iPLA2β, including the group IB secretory PLA2 (sPLA2-IB), which resides in part in insulin-secretory granules and is cosecreted with insulin (57). Although iPLA2β and sPLA2-IB both hydrolyze the sn-2 fatty acid substituents from glycerolipid substrates to yield a free fatty acid and a 2-lysophospholipid, the two enzymes reside in different compartments and have different catalytic requirements. iPLA2β is active at nanomolar to low micromolar Ca2+ concentrations that occur within the cytosol of living cells, resides predominantly in cytosol in resting cells, and undergoes subcellular redistribution to associate with membranous organelles on stimulation of β-cells with secretagogues (35, 38). In contrast, sPLA2-IB requires millimolar concentrations of Ca2+ for catalytic activity and functions as an extracellular secreted enzyme (64).
sPLA2-IB-null mice have been prepared and found to be resistant to high-fat-diet-induced obesity and insulin resistance and to exhibit reduced postprandial hyperglycemia, superior glucose tolerance, and increased insulin sensitivity compared with WT littermates (27, 32). These effects appear to be attributable to reduced absorption and blood levels of lysophospholipids, such as LPC, and to improved glucose uptake by several peripheral tissues, including liver, heart, and skeletal muscle (32). Plasma insulin levels after intraperitoneal glucose administration are slightly but not significantly lower in sPLA2-IB-null mice than in WT mice, and the improved glucose tolerance of the sPLA2-IB-null mice is thought to reflect principally improved insulin sensitivity (32).
A widely cited hypothesis is that iPLA2β is critical for synthesis of arachidonate-containing phospholipids and provides LPC acceptors for incorporating arachidonic acid into diradyl-GPC lipids (4, 64). This could be important because pancreatic islets have the highest arachidonate-containing phospholipid content of any known tissue (56), and arachidonic acid released by secretagogue-activated phospholipases is thought to promote insulin secretion (29, 31, 48, 53, 54, 74, 75). If iPLA2β overexpression increased the content of arachidonate-containing phospholipids, this might contribute to enhanced RIP-iPLA2β-TG islet secretion, but [3H]arachidonate incorporation and content of LPC and arachidonate-containing GPC lipids of WT and RIP-iPLA2β-TG islets are indistinguishable. This is consistent with data from INS-1 cells that overexpress (35) or underexpress (5) iPLA2β and from several tissues and cells from iPLA2β-null mice, including islets (9), testes (8), and peritoneal macrophages (7). These findings call into question the relevance of the iPLA2β-arachidonate incorporation 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 native macrophages (7).
In conclusion, the findings reported here with TG mice that overexpress iPLA2β in pancreatic islet β-cells support the hypothesis that iPLA2β plays a role in amplifying insulin secretion and indicate that this is relevant to the physiology of glucose homeostasis in living animals.
GRANTS
This work was supported by United States Public Health Service Grants R37-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 grant DRTC DK-20595. D. A. Jacobson was supported in part by a postdoctoral fellowship in β-cell research from Takeda Pharmaceuticals North America.
ACKNOWLEDGMENTS
We thank Sheng Zhang and Min Tan for excellent technical assistance, Dr. Sasanka Ramanadham for a critical reading of the manuscript, and Dr. Richard Gross for iPLA2β antibody 506. We also gratefully acknowledge O. Pongs, S. A. Goldstein, L. E. Fridlyand, J. P. Lopez, and N. A. Tamarina for helpful discussions and suggestions and F. Mendez and S. Eames for technical expertise and assistance.
FOOTNOTES
Address for reprint requests and other correspondence: J. Turk, Washington Univ. School of Medicine, Box 8127, 660 S. Euclid Ave., St. Louis, MO 63110 (e-mail: jturk{at}wustl.edu)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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) and WT (
) mice of ages 3–48 wk with free access to water and chow were weighed between 9:00 and 10:00 AM on a top-loading balance. Mean values are displayed, and SD are indicated (n = 44–71). None of displayed values differed significantly between genotypes.
