Studies of insulin secretory responses and of arachidonic acid incorporation into phospholipids of stably transfected insulinoma cells that overexpress group VIA phospholipase A2 (iPLA2beta ) indicate a signaling rather than a housekeeping role for iPLA2beta

A cytosolic 84-kDa group VIA phospholipase A(2) (iPLA(2)beta) that does not require Ca(2+) for catalysis has been cloned from several sources, including rat and human pancreatic islet beta-cells and murine P388D1 cells. Many potential iPLA(2)beta functions have been proposed, including a signaling role in beta-cell insulin secretion and a role in generating lysophosphatidylcholine acceptors for arachidonic acid incorporation into P388D1 cell phosphatidylcholine (PC). Proposals for iPLA(2)beta function rest in part on effects of inhibiting iPLA(2)beta activity with a bromoenol lactone (BEL) suicide substrate, but BEL also inhibits phosphatidate phosphohydrolase-1 and a group VIB phospholipase A(2). Manipulation of iPLA(2)beta expression by molecular biologic means is an alternative approach to study iPLA(2)beta functions, and we have used a retroviral construct containing iPLA(2)beta cDNA to prepare two INS-1 insulinoma cell clonal lines that stably overexpress iPLA(2)beta. Compared with parental INS-1 cells or cells transfected with empty vector, both iPLA(2)beta-overexpressing lines exhibit amplified insulin secretory responses to glucose and cAMP-elevating agents, and BEL substantially attenuates stimulated secretion. Electrospray ionization mass spectrometric analyses of arachidonic acid incorporation into INS-1 cell PC indicate that neither overexpression nor inhibition of iPLA(2)beta affects the rate or extent of this process in INS-1 cells. Immunocytofluorescence studies with antibodies directed against iPLA(2)beta indicate that cAMP-elevating agents increase perinuclear fluorescence in INS-1 cells, suggesting that iPLA(2)beta associates with nuclei. These studies are more consistent with a signaling than with a housekeeping role for iPLA(2)beta in insulin-secreting beta-cells.     

Glucose homeostasis, insulin secretion, and islet phospholipids in mice that overexpress iPLA2beta in pancreatic beta-cells and in iPLA2beta-null mice

Studies with genetically modified insulinoma cells suggest that group VIA phospholipase A(2) (iPLA(2)beta) participates in amplifying glucose-induced insulin secretion. INS-1 insulinoma cells that overexpress iPLA(2)beta, 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 iPLA(2)beta overexpression, and two characterized TG mouse lines exhibit similar phenotypes. Their pancreatic islet iPLA(2)beta expression is increased severalfold, as reflected by quantitative PCR of iPLA(2)beta mRNA, immunoblotting of iPLA(2)beta protein, and iPLA(2)beta enzymatic activity. Immunofluorescence microscopic studies of pancreatic sections confirm iPLA(2)beta overexpression in RIP-iPLA(2)beta-TG islet beta-cells without obviously perturbed islet morphology. Male RIP-iPLA(2)beta-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-iPLA(2)beta-TG mice exhibit greater amplification of glucose-induced insulin secretion by a cAMP-elevating agent than WT islets. In contrast, islets from male iPLA(2)beta-null mice exhibit blunted insulin secretion, and those mice have impaired glucose tolerance. Arachidonate incorporation into and the phospholipid composition of RIP-iPLA(2)beta-TG islets are normal, but they exhibit reduced Kv2.1 delayed rectifier current and prolonged glucose-induced action potentials and elevations of cytosolic Ca(2+) concentration that suggest a molecular mechanism for the physiological role of iPLA(2)beta to amplify insulin secretion.     

The expression and function of a group VIA calcium-independent phospholipase A2 (iPLA2beta) in beta-cells

Many cells express a Group VIA phospholipase A2, designated iPLA2beta, that does not require calcium for activation, is stimulated by ATP, and is sensitive to inhibition by a bromoenol lactone suicide substrate (BEL). Studies in various cell systems have led to the suggestion that iPLA2beta has a role in phospholipid remodeling, signal transduction, cell proliferation, and apoptosis. We have found that pancreatic islets, beta-cells, and glucose-responsive insulinoma cells express an iPLA2beta that participates in glucose-stimulated insulin secretion but is not involved in membrane phospholipid remodeling. Additionally, recent studies reveal that iPLA2beta is involved in pathways that contribute to beta-cell proliferation and apoptosis, and that various phospholipid-derived mediators are involved in these processes. Detailed characterization of the enzyme suggests that the beta-cells express multiple isoforms of iPLA2beta, and we hypothesize that these participate in different cellular functions.     

Group VIA phospholipase A2 forms a signaling complex with the calcium/calmodulin-dependent protein kinase IIbeta expressed in pancreatic islet beta-cells

Insulin-secreting pancreatic islet beta-cells express a Group VIA Ca(2+)-independent phospholipase A(2) (iPLA(2)beta) that contains a calmodulin binding site and protein interaction domains. We identified Ca(2+)/calmodulin-dependent protein kinase IIbeta (CaMKIIbeta) as a potential iPLA(2)beta-interacting protein by yeast two-hybrid screening of a cDNA library using iPLA(2)beta cDNA as bait. Cloning CaMKIIbeta cDNA from a rat islet library revealed that one dominant CaMKIIbeta isoform mRNA is expressed by adult islets and is not observed in brain or neonatal islets and that there is high conservation of the isoform expressed by rat and human beta-cells. Binary two-hybrid assays using DNA encoding this isoform as bait and iPLA(2)beta DNA as prey confirmed interaction of the enzymes, as did assays with CaMKIIbeta as prey and iPLA(2)beta bait. His-tagged CaMKIIbeta immobilized on metal affinity matrices bound iPLA(2)beta, and this did not require exogenous calmodulin and was not prevented by a calmodulin antagonist or the Ca(2+) chelator EGTA. Activities of both enzymes increased upon their association, and iPLA(2)beta reaction products reduced CaMKIIbeta activity. Both the iPLA(2)beta inhibitor bromoenol lactone and the CaMKIIbeta inhibitor KN93 reduced arachidonate release from INS-1 insulinoma cells, and both inhibit insulin secretion. CaMKIIbeta and iPLA(2)beta can be coimmunoprecipitated from INS-1 cells, and forskolin, which amplifies glucose-induced insulin secretion, increases the abundance of the immunoprecipitatable complex. These findings suggest that iPLA(2)beta and CaMKIIbeta form a signaling complex in beta-cells, consistent with reports that both enzymes participate in insulin secretion and that their expression is coinduced upon differentiation of pancreatic progenitor to endocrine progenitor cells.     

Apoptosis of insulin-secreting cells induced by endoplasmic reticulum stress is amplified by overexpression of group VIA calcium-independent phospholipase A2 (iPLA2 beta) and suppressed by inhibition of iPLA2 beta

The death of insulin-secreting beta-cells that causes type I diabetes mellitus (DM) occurs in part by apoptosis, and apoptosis also contributes to progressive beta-cell dysfunction in type II DM. Recent reports indicate that ER stress-induced apoptosis contributes to beta-cell loss in diabetes. Agents that deplete ER calcium levels induce beta-cell apoptosis by a process that is independent of increases in [Ca(2+)](i). Here we report that the SERCA inhibitor thapsigargin induces apoptosis in INS-1 insulinoma cells and that this is inhibited by a bromoenol lactone (BEL) inhibitor of group VIA calcium-independent phospholipase A(2) (iPLA(2)beta). Overexpression of iPLA(2)beta amplifies thapsigargin-induced apoptosis of INS-1 cells, and this is also suppressed by BEL. The magnitude of thapsigargin-induced INS-1 cell apoptosis correlates with the level of iPLA(2)beta expression in various cell lines, and apoptosis is associated with stimulation of iPLA(2)beta activity, perinuclear accumulation of iPLA(2)beta protein and activity, and caspase-3-catalyzed cleavage of full-length 84 kDa iPLA(2)beta to a 62 kDa product that associates with nuclei. Thapsigargin also induces ceramide accumulation in INS-1 cells, and this response is amplified in cells that overexpress iPLA(2)beta. These findings indicate that iPLA(2)beta participates in ER stress-induced apoptosis, a pathway that promotes beta-cell death in diabetes.     

Stimulation of insulin secretion and associated nuclear accumulation of iPLA(2)beta in INS-1 insulinoma cells

Accumulating evidence suggests that the cytosolic calcium-independent phospholipase A(2) (iPLA(2)beta) manifests a signaling role in insulin-secreting (INS-1) beta-cells. Earlier, we reported that insulin-secretory responses to cAMP-elevating agents are amplified in iPLA(2)beta-overexpressing INS-1 cells (Ma Z, Ramanadham S, Bohrer A, Wohltmann M, Zhang S, and Turk J. J Biol Chem 276: 13198-13208, 2001). Here, immunofluorescence, immunoaffinity, and enzymatic activity analyses are used to examine distribution of iPLA(2)beta in stimulated INS-1 cells in greater detail. Overexpression of iPLA(2)beta in INS-1 cells leads to increased accumulation of iPLA(2)beta in the nuclear fraction. Increasing glucose concentrations alone results in modest increases in insulin secretion, relative to parental cells, and in nuclear accumulation of the iPLA(2)beta protein. In contrast, cAMP-elevating agents induce robust increases in insulin secretion and in time-dependent nuclear accumulation of iPLA(2)beta fluorescence, which is reflected by increases in nuclear iPLA(2)beta protein content and specific enzymatic activity. The stimulated effects are significantly attenuated in the presence of cell-permeable inhibitors of protein phosphorylation and glycosylation. These findings suggest that conditions that amplify insulin secretion promote translocation of beta-cell iPLA(2)beta to the nuclei, where it may serve a crucial signaling role.     

Inhibition of Ca2+-independent phospholipase A2 results in insufficient insulin secretion and impaired glucose tolerance

Islet Ca2+-independent phospholipase A2 (iPLA2) is postulated to mediate insulin secretion by releasing arachidonic acid in response to insulin secretagogues. However, the significance of iPLA2 signaling in insulin secretion in vivo remains unexplored. Here we investigated the physiological role of iPLA2 in beta-cell lines, isolated islets, and mice. We showed that small interfering RNA-specific silencing of iPLA2 expression in INS-1 cells significantly reduced insulin-secretory responses of INS-1 cells to glucose. Immunohistochemical analysis revealed that mouse islet cells expressed significantly higher levels of iPLA2 than pancreatic exocrine acinar cells. Bromoenol lactone (BEL), a selective inhibitor of iPLA2, inhibited glucose-stimulated insulin secretion from isolated mouse islets; this inhibition was overcome by exogenous arachidonic acid. We also showed that iv BEL administration to mice resulted in sustained hyperglycemia and reduced insulin levels during glucose tolerance tests. Clamp experiments demonstrated that the impaired glucose tolerance was due to insufficient insulin secretion rather than decreased insulin sensitivity. Short-term administration of BEL to mice had no effect on fasting glucose levels and caused no apparent pathological changes of islets in pancreas sections. These results unambiguously demonstrate that iPLA2 signaling plays an important role in glucose-stimulated insulin secretion under physiological conditions.     

Studies of the role of group VI phospholipase A2 in fatty acid incorporation, phospholipid remodeling, lysophosphatidylcholine generation, and secretagogue-induced arachidonic acid release in pancreatic islets and insulinoma cells.

An 84-kDa group VI phospholipase A2 (iPLA2) that does not require Ca2+ for catalysis has been cloned from Chinese hamster ovary cells, murine P388D1 cells, and pancreatic islet beta-cells. A housekeeping role for iPLA2 in generating lysophosphatidylcholine (LPC) acceptors for arachidonic acid incorporation into phosphatidylcholine (PC) has been proposed because iPLA2 inhibition reduces LPC levels and suppresses arachidonate incorporation and phospholipid remodeling in P388D1 cells. Because islet beta-cell phospholipids are enriched in arachidonate, we have examined the role of iPLA2 in arachidonate incorporation into islets and INS-1 insulinoma cells. Inhibition of iPLA2 with a bromoenol lactone (BEL) suicide substrate did not suppress and generally enhanced [3H]arachidonate incorporation into these cells in the presence or absence of extracellular calcium at varied time points and BEL concentrations. Arachidonate incorporation into islet phospholipids involved deacylation-reacylation and not de novo synthesis, as indicated by experiments with varied extracellular glucose concentrations and by examining [14C]glucose incorporation into phospholipids. BEL also inhibited islet cytosolic phosphatidate phosphohydrolase (PAPH), but the PAPH inhibitor propranolol did not affect arachidonate incorporation into islet or INS-1 cell phospholipids. Inhibition of islet iPLA2 did not alter the phospholipid head-group classes into which [3H]arachidonate was initially incorporated or its subsequent transfer from PC to other lipids. Electrospray ionization mass spectrometric measurements indicated that inhibition of INS-1 cell iPLA2 accelerated arachidonate incorporation into PC and that inhibition of islet iPLA2 reduced LPC levels by 25%, suggesting that LPC mass does not limit arachidonate incorporation into islet PC. Gas chromatography/mass spectrometry measurements indicated that BEL but not propranolol suppressed insulin secretagogue-induced hydrolysis of arachidonate from islet phospholipids. In islets and INS-1 cells, iPLA2 is thus not required for arachidonate incorporation or phospholipid remodeling and may play other roles in these cells.     

Identification of calcium-independent phospholipase A2 (iPLA2) beta,and not iPLA2gamma,as the mediator of arginine vasopressin-induced arachidonic acid release in A-10 smooth muscle cells. Enantioselective mechanism-based discrimination of mammalian iPLA2s

The agonist-stimulated release of arachidonic acid (AA) from cellular phospholipids in many cell types (e.g. myocytes, beta-cells, and neurons) has been demonstrated to be primarily mediated by calcium-independent phospholipases A(2) (iPLA(2)s) that are inhibited by the mechanism-based inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (BEL). Recently, the family of mammalian iPLA(2)s has been extended to include iPLA(2)gamma, which previously could not be pharmacologically distinguished from iPLA(2)beta. To determine whether iPLA(2)beta or iPLA(2)gamma (or both) were the enzymes responsible for arginine vasopressin (AVP)-induced AA release from A-10 cells, it became necessary to inhibit selectively iPLA(2)beta and iPLA(2)gamma in intact cells. We hypothesized that the R- and S-enantiomers of BEL would possess different inhibitory potencies for iPLA(2)beta and iPLA(2)gamma. Accordingly, racemic BEL was separated into its enantiomeric constituents by chiral high pressure liquid chromatography. Remarkably, (S)-BEL was approximately an order of magnitude more selective for iPLA(2)beta in comparison to iPLA(2)gamma. Conversely, (R)-BEL was approximately an order of magnitude more selective for iPLA(2)gamma than iPLA(2)beta. The AVP-induced liberation of AA from A-10 cells was selectively inhibited by (S)-BEL (IC(50) approximately 2 microm) but not (R)-BEL, demonstrating that the overwhelming majority of AA release is because of iPLA(2)beta and not iPLA(2)gamma activity. Furthermore, pretreatment of A-10 cells with (S)-BEL did not prevent AVP-induced MAPK phosphorylation or protein kinase C translocation. Finally, two different cell-permeable protein kinase C activators (phorbol-12-myristate-13-acetate and 1,2-dioctanoyl-sn-glycerol) could not restore the ability of A-10 cells to release AA after exposure to (S)-BEL, thus supporting the downstream role of iPLA(2)beta in AVP-induced AA release.     

Modulation of the pancreatic islet beta-cell-delayed rectifier potassium channel Kv2.1 by the polyunsaturated fatty acid arachidonate

Glucose stimulates both insulin secretion and hydrolysis of arachidonic acid (AA) esterified in membrane phospholipids of pancreatic islet beta-cells, and these processes are amplified by muscarinic agonists. Here we demonstrate that nonesterified AA regulates the biophysical activity of the pancreatic islet beta-cell-delayed rectifier channel, Kv2.1. Recordings of Kv2.1 currents from INS-1 insulinoma cells incubated with AA (5 mum) and subjected to graded degrees of depolarization exhibit a significantly shorter time-to-peak current interval than do control cells. AA causes a rapid decay and reduced peak conductance of delayed rectifier currents from INS-1 cells and from primary beta-cells isolated from mouse, rat, and human pancreatic islets. Stimulating mouse islets with AA results in a significant increase in the frequency of glucose-induced [Ca(2+)] oscillations, which is an expected effect of Kv2.1 channel blockade. Stimulation with concentrations of glucose and carbachol that accelerate hydrolysis of endogenous AA from islet phosphoplipids also results in accelerated Kv2.1 inactivation and a shorter time-to-peak current interval. Group VIA phospholipase A(2) (iPLA(2)beta) hydrolyzes beta-cell membrane phospholipids to release nonesterified fatty acids, including AA, and inhibiting iPLA(2)beta prevents the muscarinic agonist-induced accelerated Kv2.1 inactivation. Furthermore, glucose and carbachol do not significantly affect Kv2.1 inactivation in beta-cells from iPLA(2)beta(-/-) mice. Stably transfected INS-1 cells that overexpress iPLA(2)beta hydrolyze phospholipids more rapidly than control INS-1 cells and also exhibit an increase in the inactivation rate of the delayed rectifier currents. These results suggest that Kv2.1 currents could be dynamically modulated in the pancreatic islet beta-cell by phospholipase-catalyzed hydrolysis of membrane phospholipids to yield non-esterified fatty acids, such as AA, that facilitate Ca(2+) entry and insulin secretion.     

Nesfatin-1 stimulates glucagon and insulin secretion and beta cell NUCB2 is reduced in human type 2 diabetic subjects

Nesfatin-1 is a novel anorexigenic regulatory peptide. The peptide is the N-terminal part of nucleobindin 2 (NUCB2) and is expressed in brain areas regulating feeding. Outside the brain, nesfatin-1 expression has been reported in adipocytes, gastric endocrine cells and islet cells. We studied NUCB2 expression in human and rodent islets using immunocytochemistry, in situ hybridization and western blot. Furthermore, we investigated the potential influence of nesfatin-1 on secretion of insulin and glucagon in vitro and in vivo in mice and in INS-1 (832/13) cells. The impact of type 2 diabetes (T2D) and glucolipotoxicity on NUCB2 gene expression in human islets and its relationship to insulin secretory capacity and islet gene expression was studied using microarray. Nesfatin-1 immunoreactivity (IR) was abundant in human and rodent beta cells but absent in alpha, delta, PP and ghrelin cells. Importantly, in situ hybridization showed that NUCB2 mRNA is expressed in human and rat islets. Western blot analysis showed that nesfatin-1 IR represented full length NUCB2 in rodent islets. Human islet NUCB2 mRNA was reduced in T2D subjects but upregulated after culture in glucolipotoxic conditions. Furthermore, a positive correlation between NUCB2 and glucagon and insulin gene expression, as well as insulin secretory capacity, was evident. Nesfatin-1 enhanced glucagon secretion but had no effect on insulin secretion from mouse islets or INS-1 (832/13) cells. On the other hand, nesfatin-1 caused a small increase in insulin secretion and reduced glucose during IVGTT in mice. We conclude that nesfatin-1 is a novel glucagon-stimulatory peptide expressed in the beta cell and that its expression is decreased in T2D islets.     

Important role of phosphodiesterase 3B for the stimulatory action of cAMP on pancreatic beta-cell exocytosis and release of insulin

Cyclic AMP potentiates glucose-stimulated insulin release and mediates the stimulatory effects of hormones such as glucagon-like peptide 1 (GLP-1) on pancreatic beta-cells. By inhibition of cAMP-degrading phosphodiesterase (PDE) and, in particular, selective inhibition of PDE3 activity, stimulatory effects on insulin secretion have been observed. Molecular and functional information on beta-cell PDE3 is, however, scarce. To provide such information, we have studied the specific effects of the PDE3B isoform by adenovirus-mediated overexpression. In rat islets and rat insulinoma cells, approximate 10-fold overexpression of PDE3B was accompanied by a 6-8-fold increase in membrane-associated PDE3B activity. The cAMP concentration was significantly lowered in transduced cells (INS-1(832/13)), and insulin secretion in response to stimulation with high glucose (11.1 mm) was reduced by 40% (islets) and 50% (INS-1). Further, the ability of GLP-1 (100 nm) to augment glucose-stimulated insulin secretion was inhibited by approximately 30% (islets) and 70% (INS-1). Accordingly, when stimulating with cAMP, a substantial decrease (65%) in exocytotic capacity was demonstrated in patch-clamped single beta-cells. In untransduced insulinoma cells, application of the PDE3-selective inhibitor OPC3911 (10 microm) was shown to increase glucose-stimulated insulin release as well as cAMP-enhanced exocytosis. The findings suggest a significant role of PDE3B as an important regulator of insulin secretory processes.     

The group VIA calcium-independent phospholipase A2 participates in ER stress-induced INS-1 insulinoma cell apoptosis by promoting ceramide generation via hydrolysis of sphingomyelins by neutral sphingomyelinase

Beta-cell mass is regulated by a balance between beta-cell growth and beta-cell death, due to apoptosis. We previously reported that apoptosis of INS-1 insulinoma cells due to thapsigargin-induced ER stress was suppressed by inhibition of the group VIA Ca2+-independent phospholipase A2 (iPLA2beta), associated with an increased level of ceramide generation, and that the effects of ER stress were amplified in INS-1 cells in which iPLA2beta was overexpressed (OE INS-1 cells). These findings suggested that iPLA2beta and ceramides participate in ER stress-induced INS-1 cell apoptosis. Here, we address this possibility and also the source of the ceramides by examining the effects of ER stress in empty vector (V)-transfected and iPLA2beta-OE INS-1 cells using apoptosis assays and immunoblotting, quantitative PCR, and mass spectrometry analyses. ER stress induced expression of ER stress factors GRP78 and CHOP, cleavage of apoptotic factor PARP, and apoptosis in V and OE INS-1 cells. Accumulation of ceramide during ER stress was not associated with changes in mRNA levels of serine palmitoyltransferase (SPT), the rate-limiting enzyme in de novo synthesis of ceramides, but both message and protein levels of neutral sphingomyelinase (NSMase), which hydrolyzes sphingomyelins to generate ceramides, were temporally increased in the INS-1 cells. The increases in the level of NSMase expression in the ER-stressed INS-1 cells were associated with corresponding temporal elevations in ER-associated iPLA2beta protein and catalytic activity. Pretreatment with BEL inactivated iPLA2beta and prevented induction of NSMase message and protein in ER-stressed INS-1 cells. Relative to that in V INS-1 cells, the effects of ER stress were accelerated and/or amplified in the OE INS-1 cells. However, inhibition of iPLA2beta or NSMase (chemically or with siRNA) suppressed induction of NSMase message, ceramide generation, sphingomyelin hydrolysis, and apoptosis in both V and OE INS-1 cells during ER stress. In contrast, inhibition of SPT did not suppress ceramide generation or apoptosis in either V or OE INS-1 cells. These findings indicate that iPLA2beta activation participates in ER stress-induced INS-1 cell apoptosis by promoting ceramide generation via NSMase-catalyzed hydrolysis of sphingomyelins, raising the possibility that this pathway contributes to beta-cell apoptosis due to ER stress.     

Insulin secretory responses and phospholipid composition of pancreatic islets from mice that do not express Group VIA phospholipase A2 and effects of metabolic stress on glucose homeostasis

Studies involving pharmacologic or molecular biologic manipulation of Group VIA phospholipase A(2) (iPLA(2)beta) activity in pancreatic islets and insulinoma cells suggest that iPLA(2)beta participates in insulin secretion. It has also been suggested that iPLA(2)beta is a housekeeping enzyme that regulates cell 2-lysophosphatidylcholine (LPC) levels and arachidonate incorporation into phosphatidylcholine (PC). We have generated iPLA(2)beta-null mice by homologous recombination and have reported that they exhibit reduced male fertility and defective motility of spermatozoa. Here we report that pancreatic islets from iPLA(2)beta-null mice have impaired insulin secretory responses to D-glucose and forskolin. Electrospray ionization mass spectrometric analyses indicate that the abundance of arachidonate-containing PC species of islets, brain, and other tissues from iPLA(2)beta-null mice is virtually identical to that of wild-type mice, and no iPLA(2)beta mRNA was observed in any tissue from iPLA(2)beta-null mice at any age. Despite the insulin secretory abnormalities of isolated islets, fasting and fed blood glucose concentrations of iPLA(2)beta-null and wild-type mice are essentially identical under normal circumstances, but iPLA(2)beta-null mice develop more severe hyperglycemia than wild-type mice after administration of multiple low doses of the beta-cell toxin streptozotocin, suggesting an impaired islet secretory reserve. A high fat diet also induces more severe glucose intolerance in iPLA(2)beta-null mice than in wild-type mice, but PLA(2)beta-null mice have greater responsiveness to exogenous insulin than do wild-type mice fed a high fat diet. These and previous findings thus indicate that iPLA(2)beta-null mice exhibit phenotypic abnormalities in pancreatic islets in addition to testes and macrophages.     

Group VIA PLA2 (iPLA2β) is activated upstream of p38 mitogen-activated protein kinase (MAPK) in pancreatic islet β-cell signaling

Group VIA phospholipase A(2) (iPLA(2)β) in pancreatic islet β-cells participates in glucose-stimulated insulin secretion and sarco(endo)plasmic reticulum ATPase (SERCA) inhibitor-induced apoptosis, and both are attenuated by pharmacologic or genetic reductions in iPLA(2)β activity and amplified by iPLA(2)β overexpression. While exploring signaling events that occur downstream of iPLA(2)β activation, we found that p38 MAPK is activated by phosphorylation in INS-1 insulinoma cells and mouse pancreatic islets, that this increases with iPLA(2)β expression level, and that it is stimulated by the iPLA(2)β reaction product arachidonic acid. The insulin secretagogue D-glucose also stimulates β-cell p38 MAPK phosphorylation, and this is prevented by the iPLA(2)β inhibitor bromoenol lactone. Insulin secretion induced by d-glucose and forskolin is amplified by overexpressing iPLA(2)β in INS-1 cells and in mouse islets, and the p38 MAPK inhibitor PD169316 prevents both responses. The SERCA inhibitor thapsigargin also stimulates phosphorylation of both β-cell MAPK kinase isoforms and p38 MAPK, and bromoenol lactone prevents both events. Others have reported that iPLA(2)β products activate Rho family G-proteins that promote MAPK kinase activation via a mechanism inhibited by Clostridium difficile toxin B, which we find to inhibit thapsigargin-induced β-cell p38 MAPK phosphorylation. Thapsigargin-induced β-cell apoptosis and ceramide generation are also prevented by the p38 MAPK inhibitor PD169316. These observations indicate that p38 MAPK is activated downstream of iPLA(2)β in β-cells incubated with insulin secretagogues or thapsigargin, that this requires prior iPLA(2)β activation, and that p38 MAPK is involved in the β-cell functional responses of insulin secretion and apoptosis in which iPLA(2)β participates.     

Effects of stable suppression of Group VIA phospholipase A2 expression on phospholipid content and composition, insulin secretion, and proliferation of INS-1 insulinoma cells

Studies involving pharmacologic inhibition or transient reduction of Group VIA phospholipase A2 (iPLA2beta) expression have suggested that it is a housekeeping enzyme that regulates cell 2-lysophosphatidylcholine (LPC) levels, rates of arachidonate incorporation into phospholipids, and degradation of excess phosphatidylcholine (PC). In insulin-secreting islet beta-cells and some other cells, in contrast, iPLA2beta signaling functions have been proposed. Using retroviral vectors, we prepared clonal INS-1 beta-cell lines in which iPLA2beta expression is stably suppressed by small interfering RNA. Two such iPLA2beta knockdown (iPLA2beta-KD) cell lines express less than 20% of the iPLA2beta of control INS-1 cell lines. The iPLA2beta-KD INS-1 cells exhibit impaired insulin secretory responses and reduced proliferation rates. Electrospray ionization mass spectrometric analyses of PC and LPC species that accumulate in INS-1 cells cultured with arachidonic acid suggest that 18:0/20:4-glycerophosphocholine (GPC) synthesis involves sn-2 remodeling to yield 16:0/20:4-GPC and then sn-1 remodeling via a 1-lyso/20:4-GPC intermediate. Electrospray ionization mass spectrometric analyses also indicate that the PC and LPC content and composition of iPLA2beta-KD and control INS-1 cells are nearly identical, as are the rates of arachidonate incorporation into PC and the composition and remodeling of other phospholipid classes. These findings indicate that iPLA2beta plays signaling or effector roles in beta-cell secretion and proliferation but that stable suppression of its expression does not affect beta-cell GPC lipid content or composition even under conditions in which LPC is being actively consumed by conversion to PC. This calls into question the generality of proposed housekeeping functions for iPLA2beta in PC homeostasis and remodeling.     

Glucose transporter isotypes switch in T-antigen-transformed pancreatic beta cells growing in culture and in mice.

High-level expression of the low-Km glucose transporter isoform GLUT-1 is characteristic of many cultured tumor and oncogene-transformed cells. In this study, we tested whether induction of GLUT-1 occurs in tumors in vivo. Normal mouse beta islet cells express the high-Km (approximately 20 mM) glucose transporter isoform GLUT-2 but not the low-Km (1 to 3 mM) GLUT-1. In contrast, a beta cell line derived from an insulinoma arising in a transgenic mouse harboring an insulin-promoted simian virus 40 T-antigen oncogene (beta TC3) expressed very low levels of GLUT-2 but high levels of GLUT-1. GLUT-1 protein was not detectable on the plasma membrane of islets or tumors of the transgenic mice but was induced in high amounts when the tumor-derived beta TC3 cells were grown in tissue culture. GLUT-1 expression in secondary tumors formed after injection of beta TC3 cells into mice was reduced. Thus, high-level expression of GLUT-1 in these tumor cells is characteristic of culture conditions and is not induced by the oncogenic transformation; indeed, overnight culture of normal pancreatic islets causes induction of GLUT-1. We also investigated the relationship between expression of the different glucose transporter isoforms by islet and tumor cells and induction of insulin secretion by glucose. Prehyperplastic transgenic islet cells that expressed normal levels of GLUT-2 and no detectable GLUT-1 exhibited an increased sensitivity to glucose, as evidenced by maximal insulin secretion at lower glucose concentrations, compared with that exhibited by normal islets. Further, hyperplastic islets and primary and secondary tumors expressed low levels of GLUT-2 and no detectable GLUT-1 on the plasma membrane; these cells exhibited high basal insulin secretion and responded poorly to an increase in extracellular glucose. Thus, abnormal glucose-induced secretion of insulin in prehyperplastic islets in mice was independent of changes in GLUT-2 expression and did not require induction of GLUT-1 expression.