Shunzhong Bao

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 A2 (iPLA2β) activity in pancreatic islets and insulinoma cells suggest that iPLA2β participates in insulin secretion. It has also been suggested that iPLA2β is a housekeeping enzyme that regulates cell 2-lysophosphatidylcholine (LPC) levels and arachidonate incorporation into phosphatidylcholine (PC). We have generated iPLA2β-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 iPLA2β-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 iPLA2β-null mice is virtually identical to that of wild-type mice, and no iPLA2β mRNA was observed in any tissue from iPLA2β-null mice at any age. Despite the insulin secretory abnormalities of isolated islets, fasting and fed blood glucose concentrations of iPLA2β-null and wild-type mice are essentially identical under normal circumstances, but iPLA2β-null mice develop more severe hyperglycemia than wild-type mice after administration of multiple low doses of the β-cell toxin streptozotocin, suggesting an impaired islet secretory reserve. A high fat diet also induces more severe glucose intolerance in iPLA2β-null mice than in wild-type mice, but PLA2β-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 iPLA2β-null mice exhibit phenotypic abnormalities in pancreatic islets in addition to testes and macrophages.

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 (iPLA2β) 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 β-cells and some other cells, in contrast, iPLA2β signaling functions have been proposed. Using retroviral vectors, we prepared clonal INS-1 β-cell lines in which iPLA2β expression is stably suppressed by small interfering RNA. Two such iPLA2β knockdown (iPLA2β-KD) cell lines express less than 20% of the iPLA2β of control INS-1 cell lines. The iPLA2β-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 iPLA2β-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 iPLA2β plays signaling or effector roles in β-cell secretion and proliferation but that stable suppression of its expression does not affect β-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 iPLA2β in PC homeostasis and remodeling.

Modulation of the Pancreatic Islet β-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 β-cells, and these processes are amplified by muscarinic agonists. Here we demonstrate that nonesterified AA regulates the biophysical activity of the pancreatic islet β-cell-delayed rectifier channel, Kv2.1. Recordings of Kv2.1 currents from INS-1 insulinoma cells incubated with AA (5 μm) 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 β-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 [Ca2+] 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 A2 (iPLA2β) hydrolyzes β-cell membrane phospholipids to release nonesterified fatty acids, including AA, and inhibiting iPLA2β prevents the muscarinic agonist-induced accelerated Kv2.1 inactivation. Furthermore, glucose and carbachol do not significantly affect Kv2.1 inactivation in β-cells from iPLA2β-/- mice. Stably transfected INS-1 cells that overexpress iPLA2β 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 β-cell by phospholipase-catalyzed hydrolysis of membrane phospholipids to yield non-esterified fatty acids, such as AA, that facilitate Ca2+ entry and insulin secretion.

Attenuated Free Cholesterol Loading-induced Apoptosis but Preserved Phospholipid Composition of Peritoneal Macrophages from Mice That Do Not Express Group VIA Phospholipase A2

Mouse macrophages undergo ER stress and apoptosis upon free cholesterol loading (FCL). We recently generated iPLA2β-null mice, and here we demonstrate that iPLA2β-null macrophages have reduced sensitivity to FCL-induced apoptosis, although they and wild-type (WT) cells exhibit similar increases in the transcriptional regulator CHOP. iPLA2β-null macrophages are also less sensitive to apoptosis induced by the sarcoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin and the scavenger receptor A ligand fucoidan, and restoring iPLA2βexpression with recombinant adenovirus increases apoptosis toward WT levels. WT and iPLA2β-null macrophages incorporate [3H]arachidonic acid ([3H]AA]) into glycerophosphocholine lipids equally rapidly and exhibit identical zymosan-induced, cPLA2α-catalyzed [3H]AA release. In contrast, although WT macrophages exhibit robust [3H]AA release upon FCL, this is attenuated in iPLA2β-null macrophages and increases toward WT levels upon restoring iPLA2β expression. Recent reports indicate that iPLA2β modulates mitochondrial cytochrome c release, and we find that thapsigargin and fucoidan induce mitochondrial phospholipid loss and cytochrome c release into WT macrophage cytosol and that these events are blunted in iPLA2β-null cells. Immunoblotting studies indicate that iPLA2β associates with mitochondria in macrophages subjected to ER stress. AA incorporation into glycerophosphocholine lipids is unimpaired in iPLA2β-null macrophages upon electrospray ionization-tandem mass spectrometry analyses, and their complex lipid composition is similar to WT cells. These findings suggest that iPLA2β participates in ER stress-induced macrophage apoptosis caused by FCL or thapsigargin but that deletion of iPLA2β does not impair macrophage arachidonate incorporation or phospholipid composition.

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

Insulin-secreting pancreatic islet β-cells express a Group VIA Ca2+-independent phospholipase A2 (iPLA2β) that contains a calmodulin binding site and protein interaction domains. We identified Ca2+/calmodulindependent protein kinase IIβ (CaMKIIβ) as a potential iPLA2β-interacting protein by yeast two-hybrid screening of a cDNA library using iPLA2β cDNA as bait. Cloning CaMKIIβ cDNA from a rat islet library revealed that one dominant CaMKIIβ 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 β-cells. Binary two-hybrid assays using DNA encoding this isoform as bait and iPLA2β DNA as prey confirmed interaction of the enzymes, as did assays with CaMKIIβ as prey and iPLA2β bait. His-tagged CaMKIIβ immobilized on metal affinity matrices bound iPLA2β, and this did not require exogenous calmodulin and was not prevented by a calmodulin antagonist or the Ca2+ chelator EGTA. Activities of both enzymes increased upon their association, and iPLA2β reaction products reduced CaMKIIβ activity. Both the iPLA2β inhibitor bromoenol lactone and the CaMKIIβ inhibitor KN93 reduced arachidonate release from INS-1 insulinoma cells, and both inhibit insulin secretion. CaMKIIβ and iPLA2β 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 iPLA2β and CaMKIIβ form a signaling complex in β-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.

Mice deficient in Group VIB phospholipase A2 (iPLA2γ) exhibit relative resistance to obesity and metabolic abnormalities induced by a Western diet

Phospholipases A(2) (PLA(2)) play important roles in metabolic processes, and the Group VI PLA(2) family is comprised of intracellular enzymes that do not require Ca(2+) for catalysis. Mice deficient in Group VIA PLA(2) (iPLA(2)beta) develop more severe glucose intolerance than wild-type (WT) mice in response to dietary stress. Group VIB PLA(2) (iPLA(2)gamma) is a related enzyme distributed in membranous organelles, including mitochondria, and iPLA(2)gamma knockout (KO) mice exhibit altered mitochondrial morphology and function. We have compared metabolic responses of iPLA(2)gamma-KO and WT mice fed a Western diet (WD) with a high fat content. We find that KO mice are resistant to WD-induced increases in body weight and adiposity and in blood levels of cholesterol, glucose, and insulin, even though WT and KO mice exhibit similar food consumption and dietary fat digestion and absorption. KO mice are also relatively resistant to WD-induced insulin resistance, glucose intolerance, and altered patterns of fat vs. carbohydrate fuel utilization. KO skeletal muscle exhibits impaired mitochondrial beta-oxidation of fatty acids, as reflected by accumulation of larger amounts of long-chain acylcarnitine (LCAC) species in KO muscle and liver compared with WT in response to WD feeding. This is associated with increased urinary excretion of LCAC and much reduced deposition of triacylglycerols in liver by WD-fed KO compared with WT mice. The iPLA(2)gamma-deficient genotype thus results in a phenotype characterized by impaired mitochondrial oxidation of fatty acids and relative resistance to the metabolic abnormalities induced by WD.

Group VIA PLA2 (iPLA2β) Is Activated Upstream of p38 Mitogen-activated Protein Kinase (MAPK) in Pancreatic Islet β-Cell Signaling

Background: Group VIA Phospholipase A2 (iPLA2β) is activated in β-cell signaling. Results: β-Cell p38 MAPK is activated by an iPLA2β-dependent mechanism and participates in insulin secretion and apoptosis. Conclusion: p38 MAPK is a downstream effector of iPLA2β activation in β-cells. Significance: Understanding how insulin secretion is impaired and β-cell apoptosis occurs could suggest therapeutic strategies for type 2 diabetes mellitus. Group VIA phospholipase A2 (iPLA2β) 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 iPLA2β activity and amplified by iPLA2β overexpression. While exploring signaling events that occur downstream of iPLA2β activation, we found that p38 MAPK is activated by phosphorylation in INS-1 insulinoma cells and mouse pancreatic islets, that this increases with iPLA2β expression level, and that it is stimulated by the iPLA2β reaction product arachidonic acid. The insulin secretagogue d-glucose also stimulates β-cell p38 MAPK phosphorylation, and this is prevented by the iPLA2β inhibitor bromoenol lactone. Insulin secretion induced by d-glucose and forskolin is amplified by overexpressing iPLA2β 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 iPLA2β 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 iPLA2β in β-cells incubated with insulin secretagogues or thapsigargin, that this requires prior iPLA2β activation, and that p38 MAPK is involved in the β-cell functional responses of insulin secretion and apoptosis in which iPLA2β participates.

Evidence for Proteolytic Processing and Stimulated Organelle Redistribution of iPLA2β

Over the past decade, important roles for the 84-88kDa Group VIA Ca(2+)-independent phospholipase A(2) (iPLA(2)beta) in various organs have been described. We demonstrated that iPLA(2)beta participates in insulin secretion, insulinoma cells and native pancreatic islets express full-length and truncated isoforms of iPLA(2)beta, and certain stimuli promote perinuclear localization of iPLA(2)beta. To gain a better understanding of its mobilization, iPLA(2)beta was expressed in INS-1 cells as a fusion protein with EGFP, enabling detection of subcellular localization of iPLA(2)beta by monitoring EGFP fluorescence. Cells stably-transfected with fusion protein expressed nearly 5-fold higher catalytic iPLA(2)beta activity than control cells transfected with EGFP cDNA alone, indicating that co-expression of EGFP does not interfere with manifestation of iPLA(2)beta activity. Dual fluorescence monitoring of EGFP and organelle Trackers combined with immunoblotting analyses revealed expression of truncated iPLA(2)beta isoforms in separate subcellular organelles. Exposure to secretagogues and induction of ER stress are known to activate iPLA(2)beta in beta-cells and we find here that these stimuli promote differential localization of iPLA(2)beta in subcellular organelles. Further, mass spectrometric analyses identified iPLA(2)beta variants from which N-terminal residues were removed. Collectively, these findings provide evidence for endogenous proteolytic processing of iPLA(2)beta and redistribution of iPLA(2)beta variants in subcellular compartments. It might be proposed that in vivo processing of iPLA(2)beta facilitates its participation in multiple biological processes.

Calcium-Independent Phospholipase A2β Is Dispensable in Inflammasome Activation and Its Inhibition by Bromoenol Lactone

Calcium-independent phospholipase A2 (iPLA2) has been suggested to play an important role in the activation of caspase-1 induced by lipopolysaccharides (LPS). Here, we used pharmacological and genetic approaches to study the role of iPLA2 in the activation of caspase-1. Bromoenol lactone (BEL), an inhibitor that was originally used to support a role for iPLA2 in the secretion of IL-1β, prevented caspase-1 activation induced by LPS and ATP as described, and also activation triggered by Salmonella infection and cytosolic flagellin, which rely on the Nlrc4 inflammasome. Analysis of BEL enantiomers showed that the S-BEL form was more effective than R-BEL in inhibiting the inflammasome, suggesting a role for iPLA2β. However, caspase-1 activation and IL-1β secretion and their inhibition by BEL were unimpaired in macrophages deficient in iPLA2β. BEL was originally identified as an inhibitor of serine proteases. Consistent with the latter, the serine proteases inhibitors TPCK, TLCK and AAF-cmk prevented the activation of the Nlrc4 and Nlrp3 inflammasomes while pan-cathepsin inhibitors were ineffective. These results indicate that iPLA2β is not critical for caspase-1 activation as currently proposed. Instead, the results suggest that serine protease(s) targeted by BEL may play a critical role in the activation of the inflammasome triggered by microbial stimuli.

Group VIA Phospholipase A2 Mediates Enhanced Macrophage Migration in Diabetes Mellitus by Increasing Expression of Nicotinamide Adenine Dinucleotide Phosphate Oxidase 4

Objective—We previously demonstrated that nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) mediates increased monocyte priming and chemotaxis under conditions of diabetic metabolic stress, and emerging data indicate that group VIA phospholipase A2 (iPLA2&bgr;) also participates in regulating monocyte chemotaxis. Here, we examined relationships between iPLA2&bgr; expression and Nox4 action in mouse peritoneal macrophages subjected to diabetic metabolic stress. Approach and Results—Increased iPLA2&bgr; expression and activity were observed in macrophages from low-density lipoprotein receptor knockout mice that were fed a high-fat diet, and this was associated with time-dependent increases in atherosclerotic lesion size and macrophage content. Incubating macrophages with 30 mmol/L D-glucose, 100 &mgr;g/mL low-density lipoprotein, or both (D-glucose+low-density lipoprotein) induced a robust increase in iPLA2&bgr; expression and activity and in cell migration in response to monocyte chemoattractant protein-1. The increases in iPLA2&bgr; activity and cell migration were prevented by a bromoenol lactone iPLA2&bgr; suicide inhibitor or an iPLA2&bgr; antisense oligonucleotide. Incubating macrophages under conditions that mimic diabetic metabolic stress ex vivo resulted in increased Nox4 expression and activity and hydrogen peroxide generation compared with controls. Bromoenol lactone prevented those effects without affecting Nox2 expression. Nox4 inhibition eliminated diabetic metabolic stress–induced acceleration of macrophage migration. Lysophosphatidic acid restored Nox4 expression, hydrogen peroxide generation, and migration to bromoenol lactone–treated cells, and a lysophosphatidic acid receptor antagonist abrogated iPLA2&bgr;-mediated increases in Nox4 expression. Conclusions—Taken together, these observations identify iPLA2&bgr; and lysophosphatidic acid derived from its action as critical in regulating macrophage Nox4 activity and migration in the diabetic state in vivo and under similar conditions ex vivo.