Christopher M. Jenkins

Identification of Calcium-independent Phospholipase A2 (iPLA2) β, and Not iPLA2γ, 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, β-cells, and neurons) has been demonstrated to be primarily mediated by calcium-independent phospholipases A2 (iPLA2s) that are inhibited by the mechanism-based inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (BEL). Recently, the family of mammalian iPLA2s has been extended to include iPLA2γ, which previously could not be pharmacologically distinguished from iPLA2β. To determine whether iPLA2β or iPLA2γ (or both) were the enzymes responsible for arginine vasopressin (AVP)-induced AA release from A-10 cells, it became necessary to inhibit selectively iPLA2β and iPLA2γ in intact cells. We hypothesized that the R- and S-enantiomers of BEL would possess different inhibitory potencies for iPLA2β and iPLA2γ. 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 iPLA2β in comparison to iPLA2γ. Conversely, (R)-BEL was approximately an order of magnitude more selective for iPLA2γ than iPLA2β. The AVP-induced liberation of AA from A-10 cells was selectively inhibited by (S)-BEL (IC50 ∼2 μm) but not (R)-BEL, demonstrating that the overwhelming majority of AA release is because of iPLA2β and not iPLA2γ 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 iPLA2β in AVP-induced AA release.

Eicosanoid signalling pathways in the heart

Myocardial phospholipids serve as primary reservoirs of arachidonic acid (AA), which is liberated through the rate-determining hydrolytic action of cardiac phospholipases A2 (PLA2s). A predominant PLA2 in myocardium is calcium-independent phospholipase A2beta (iPLA2beta), which, through its calmodulin (CaM) and ATP-binding domains, is regulated by alterations in local cellular Ca2+ concentrations and cardiac bioenergetic status, respectively. Importantly, iPLA2beta has been demonstrated to be activated by ischaemia through elevation of the concentration of myocardial fatty acyl-CoA, which abrogates Ca2+/CaM-mediated inhibition of iPLA2beta. AA released by PLA2-catalysed hydrolysis of phospholipids serves as a precursor for eicosanoids generated by pathways dependent on cyclooxygenases (COX), lipoxygenases (LOX), and cytochromes P450 (CYP). Eicosanoids initiate and propagate diverse signalling cascades, primarily through their interaction with cellular receptors and ion channels. However, during pathologic states such as ischaemia or congestive heart failure, eicosanoids contribute to multiple maladaptive changes including inflammation, alterations of cellular growth programmes, and activation of multiple transcriptional events leading to the deleterious sequelae of these pathologic states. This review summarizes the central roles of myocardial PLA(2)s in eicosanoid signalling in the heart, the major COX, LOX, and CYP pathways of eicosanoid generation in the myocardium, and the effects of important eicosanoids on receptor-, ion channel-, and transcription-mediated processes that facilitate cardiac hypertrophy, mediate ischaemic preconditioning, and precipitate arrhythmogenesis in response to pathologic stimuli.

Genetic Ablation of Calcium-independent Phospholipase A2γ Leads to Alterations in Mitochondrial Lipid Metabolism and Function Resulting in a Deficient Mitochondrial Bioenergetic Phenotype

Previously, we identified a novel calcium-independent phospholipase, designated calcium-independent phospholipase A2 γ (iPLA2γ), which possesses dual mitochondrial and peroxisomal subcellular localization signals. To identify the roles of iPLA2γ in cellular bioenergetics, we generated mice null for the iPLA2γ gene by eliminating the active site of the enzyme through homologous recombination. Mice null for iPLA2γ display multiple bioenergetic dysfunctional phenotypes, including 1) growth retardation, 2) cold intolerance, 3) reduced exercise endurance, 4) greatly increased mortality from cardiac stress after transverse aortic constriction, 5) abnormal mitochondrial function with a 65% decrease in ascorbate-induced Complex IV-mediated oxygen consumption, and 6) a reduction in myocardial cardiolipin content accompanied by an altered cardiolipin molecular species composition. We conclude that iPLA2γ is essential for maintaining efficient bioenergetic mitochondrial function through tailoring mitochondrial membrane lipid metabolism and composition.

Identification of the Calmodulin-binding Domain of Recombinant Calcium-independent Phospholipase A2β IMPLICATIONS FOR STRUCTURE AND FUNCTION

Calcium-independent phospholipase A2 (iPLA2) is the major phospholipase A2 activity in many cell types, and at least one isoform of this enzyme class is physically and functionally coupled to calmodulin (CaM) in a reversible calcium-dependent fashion. To identify the domain in recombinant iPLA2β (riPLA2β) underlying this interaction, multiple techniques were employed. First, we identified calcium-activated CaM induced alterations in the kinetics of proteolytic fragment generation during limited trypsinolysis (i.e. CaM footprinting). Tryptic digests of riPLA2β (83 kDa) in the presence of EGTA alone, Ca+2 alone, or EGTA and CaM together resulted in the production of a major 68-kDa protein whose kinetic rate of formation was specifically attenuated in incubations containing CaM and Ca+2 together. Western blotting utilizing antibodies directed against either the N- or C-terminal regions of riPLA2β indicated the specific protection of riPLA2β by calcium-activated CaM at a cleavage site ≈15 kDa from the C terminus. Moreover, calcium-activated calmodulin increased the kinetic rate of tryptic cleavage near the active site of riPLA2β. Second, functional characterization of products from these partial tryptic digests demonstrated that ≈90% of the 68-kDa riPLA2β tryptic product (i.e. lacking the 15-kDa C-terminus) did not bind to a CaM affinity matrix in the presence of Ca2+, although >95% of the noncleaved riPLA2β as well as a 40-kDa C-terminal peptide bound tightly under these conditions. Third, when purified riPLA2β was subjected to exhaustive trypsinolysis followed by ternary complex CaM affinity chromatography, a unique tryptic peptide (694AWSEMVGIQYFR705) within the 15-kDa C-terminal fragment was identified by RP-HPLC, which bound to CaM-agarose in the presence but not the absence of calcium ion. Fourth, fluorescence energy transfer experiments demonstrated that this peptide (694) bound to dansyl-calmodulin in a calcium-dependent fashion. Collectively, these results identify multiple contact points in the 15-kDa C terminus as being the major but not necessarily the only binding site responsible for the calcium-dependent regulation of iPLA2β by CaM.

Genetic Ablation of Calcium-independent Phospholipase A2γ Leads to Alterations in Hippocampal Cardiolipin Content and Molecular Species Distribution, Mitochondrial Degeneration, Autophagy, and Cognitive Dysfunction

Genetic ablation of calcium-independent phospholipase A2γ (iPLA2γ) results in profound alterations in hippocampal phospholipid metabolism and mitochondrial phospholipid homeostasis resulting in enlarged and degenerating mitochondria leading to autophagy and cognitive dysfunction. Shotgun lipidomics demonstrated multiple alterations in hippocampal lipid metabolism in iPLA2γ−/− mice including: 1) a markedly elevated hippocampal cardiolipin content with an altered molecular species composition characterized by a shift to shorter chain length molecular species; 2) alterations in both choline and ethanolamine glycerophospholipids, including a decreased plasmenylethanolamine content; 3) increased oxidized phosphatidylethanolamine molecular species; and 4) an increased content of ceramides. Electron microscopic examination demonstrated the presence of enlarged heteromorphic lamellar structures undergoing degeneration accompanied by the presence of ubiquitin positive spheroid inclusion bodies. Purification of these enlarged heteromorphic lamellar structures by buoyant density centrifugation and subsequent SDS-PAGE and proteomics identified them as degenerating mitochondria. Collectively, these results identify the obligatory role of iPLA2γ in neuronal mitochondrial lipid metabolism and membrane structure demonstrating that iPLA2γ loss of function results in a mitochondrial neurodegenerative disorder characterized by degenerating mitochondria, autophagy, and cognitive dysfunction.

CD36 Protein Is Involved in Store-operated Calcium Flux, Phospholipase A2 Activation, and Production of Prostaglandin E2

The scavenger receptor FAT/CD36 contributes to the inflammation associated with diabetes, atherosclerosis, thrombosis, and Alzheimer disease. Underlying mechanisms include CD36 promotion of oxidative stress and its signaling to stress kinases. Here we document an additional mechanism for the role of CD36 in inflammation. CD36 regulates membrane calcium influx in response to endoplasmic reticulum (ER) stress, release of arachidonic acid (AA) from cellular membranes by cytoplasmic phospholipase A2α (cPLA2α) and contributes to the generation of proinflammatory eicosanoids. CHO cells stably expressing human CD36 released severalfold more AA and prostaglandin E2 (PGE2), a major product of AA metabolism by cyclooxygenases, in response to thapsigargin-induced ER stress as compared with control cells. Calcium influx after ER calcium release resulted in phosphorylation of cPLA2 and its translocation to membranes in a CD36-dependent manner. Peritoneal macrophages from CD36−/− mice exhibited diminished calcium transients and reduced AA release after thapsigargin or UTP treatment with decreased ERK1/2 and cPLA2 phosphorylation. However, PGE2 production was unexpectedly enhanced in CD36−/− macrophages, which probably resulted from a large induction of cyclooxygenase 2 mRNA and protein. The data demonstrate participation of CD36 in membrane calcium influx in response to ER stress or purinergic receptor stimulation resulting in AA liberation for PGE2 formation. Collectively, these results identify a mechanism contributing to the pleiotropic proinflammatory effects of CD36 and suggest that its targeted inhibition may reduce the acute inflammatory response.

Genetic Ablation of Calcium-independent Phospholipase A2γ Prevents Obesity and Insulin Resistance during High Fat Feeding by Mitochondrial Uncoupling and Increased Adipocyte Fatty Acid Oxidation

Phospholipases are critical enzyme mediators participating in many aspects of cellular function through modulating the generation of lipid 2nd messengers, membrane physical properties, and cellular bioenergetics. Here, we demonstrate that mice null for calcium-independent phospholipase A2γ (iPLA2γ−/−) are completely resistant to high fat diet-induced weight gain, adipocyte hypertrophy, hyperinsulinemia, and insulin resistance, which occur in iPLA2γ+/+ mice after high fat feeding. Notably, iPLA2γ−/− mice were lean, demonstrated abdominal lipodystrophy, and remained insulin-sensitive despite having a marked impairment in glucose-stimulated insulin secretion after high fat feeding. Respirometry of adipocyte explants from iPLA2γ−/− mice identified increased rates of oxidation of multiple different substrates in comparison with adipocyte explants from wild-type littermates. Shotgun lipidomics of adipose tissue from wild-type mice demonstrated the anticipated 2-fold increase in triglyceride content after high fat feeding. In sharp contrast, the adipocyte triglyceride content was identical in iPLA2γ−/− mice fed either a standard diet or a high fat diet. Respirometry of skeletal muscle mitochondria from iPLA2γ−/− mice demonstrated marked decreases in state 3 respiration using multiple substrates whose metabolism was uncoupled from ATP production. Shotgun lipidomics of skeletal muscle revealed a decreased content of cardiolipin with an altered molecular species composition thereby identifying the mechanism underlying mitochondrial uncoupling in the iPLA2γ−/− mouse. Collectively, these results identify iPLA2γ as an obligatory upstream enzyme that is necessary for efficient electron transport chain coupling and energy production through its participation in the alterations of cellular bioenergetics that promote the development of the metabolic syndrome.

Small Interfering RNA Knockdown of Calcium-independent Phospholipases A2 β or γ Inhibits the Hormone-induced Differentiation of 3T3-L1 Preadipocytes

Alterations in lipid secondary messenger generation and lipid metabolic flux are essential in promoting the differentiation of adipocytes. To determine whether specific subtypes of intracellular phospholipases A2 (PLA2s) facilitate hormone-induced differentiation of 3T3-L1 cells into adipocytes, we examined alterations in the mRNA level, protein mass, and activity of three previously characterized mammalian intracellular PLA2s. Hormone-induced differentiation of 3T3-L1 cells resulted in 7.3 ± 0.5- and 7.4 ± 1.4-fold increases of mRNA encoding the calcium-independent phospholipases, iPLA2β and iPLA2γ, respectively. In contrast, the temporally coordinated loss of at least 90% of cPLA2α mRNA was manifest. Western analysis demonstrated the near absence of both iPLA2β and iPLA2γ protein mass in resting 3T3-L1 cells that increased dramatically during differentiation. In vitro measurement of PLA2 activities demonstrated an increase in both iPLA2β and iPLA2γ activities that were discriminated using the chiral mechanism based inhibitors (S)- and (R)-BEL, respectively. Remarkably, treatment of 3T3-L1 cells with small interfering RNA directed against either iPLA2β or iPLA2γ prevented hormone-induced differentiation. Moreover, analysis of the temporally programmed expression of transcription factors demonstrated that the small interfering RNA knockdown of iPLA2β or iPLA2γ resulted in down-regulation of the expression of peroxisome proliferator-activated receptor γ and the CCAAT enhancer-binding protein α (C/EBPα). No alterations in the expression of the early stage transcription factors C/EBPβ and C/EBPδ were observed. Collectively, these results demonstrate prominent alterations in intracellular PLA2s during 3T3-L1 cell differentiation into adipocytes and identify the requirement of iPLA2β and iPLA2γ for the adipogenic program that drives resting 3T3-L1 cells into adipocytes after hormone stimulation.

Dramatic Accumulation of Triglycerides and Precipitation of Cardiac Hemodynamic Dysfunction during Brief Caloric Restriction in Transgenic Myocardium Expressing Human Calcium-independent Phospholipase A2γ

Previously, we identified calcium-independent phospholipase A2γ (iPLA2γ) with multiple translation initiation sites and dual mitochondrial and peroxisomal localization motifs. To determine the role of iPLA2γ in integrating lipid and energy metabolism, we generated transgenic mice containing the α-myosin heavy chain promoter (αMHC) placed proximally to the human iPLA2γ coding sequence that resulted in cardiac myocyte-restricted expression of iPLA2γ (TGiPLA2γ). TGiPLA2γ mice possessed multiple phenotypes including: 1) a dramatic ∼35% reduction in myocardial phospholipid mass in both the fed and mildly fasted states; 2) a marked accumulation of triglycerides during brief caloric restriction that represented 50% of total myocardial lipid mass; and 3) acute fasting-induced hemodynamic dysfunction. Biochemical characterization of the TGiPLA2γ protein expressed in cardiac myocytes demonstrated over 25 distinct isoforms by two-dimensional SDS-PAGE Western analysis. Immunohistochemistry identified iPLA2γ in the peroxisomal and mitochondrial compartments in both wild type and transgenic myocardium. Electron microscopy revealed the presence of loosely packed and disorganized mitochondrial cristae in TGiPLA2γ mice that were accompanied by defects in mitochondrial function. Moreover, markedly elevated levels of 1-hydroxyl-2-arachidonoyl-sn-glycero-3-phosphocholine and 1-hydroxyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine were prominent in the TGiPLA2γ myocardium identifying the production of signaling metabolites by this enzyme in vivo. Collectively, these results identified the participation of iPLA2γ in the remarkable lipid plasticity of myocardium, its role in generating signaling metabolites, and its prominent effects in modulating energy storage and utilization in myocardium in different metabolic contexts.

Reversible High Affinity Inhibition of Phosphofructokinase-1 by Acyl-CoA A MECHANISM INTEGRATING GLYCOLYTIC FLUX WITH LIPID METABOLISM

The enzyme phosphofructokinase-1 (PFK-1) catalyzes the first committed step of glycolysis and is regulated by a complex array of allosteric effectors that integrate glycolytic flux with cellular bioenergetics. Here, we demonstrate the direct, potent, and reversible inhibition of purified rabbit muscle PFK-1 by low micromolar concentrations of long chain fatty acyl-CoAs (apparent Ki ∼1 μm). In sharp contrast, short chain acyl-CoAs, palmitoylcarnitine, and palmitic acid in the presence of CoASH were without effect. Remarkably, MgAMP and MgADP but not MgATP protected PFK-1 against inhibition by palmitoyl-CoA indicating that acyl-CoAs regulate PFK-1 activity in concert with cellular high energy phosphate status. Furthermore, incubation of PFK-1 with [1-14C]palmitoyl-CoA resulted in robust acylation of the enzyme that was reversible by incubation with acyl-protein thioesterase-1 (APT1). Importantly, APT1 reversed palmitoyl-CoA-mediated inhibition of PFK-1 activity. Mass spectrometric analyses of palmitoylated PFK-1 revealed four sites of acylation, including Cys-114, Cys-170, Cys-351, and Cys-577. PFK-1 in both skeletal muscle extracts and in purified form was inhibited by S-hexadecyl-CoA, a nonhydrolyzable palmitoyl-CoA analog, demonstrating that covalent acylation of PFK-1 was not required for inhibition. Tryptic footprinting suggested that S-hexadecyl-CoA induced a conformational change in PFK-1. Both palmitoyl-CoA and S-hexadecyl-CoA increased the association of PFK-1 with Ca2+/calmodulin, which attenuated the binding of palmitoylated PFK-1 to membrane vesicles. Collectively, these results demonstrate that fatty acyl-CoA modulates phosphofructokinase activity through both covalent and noncovalent interactions to regulate glycolytic flux and enzyme membrane localization via the branch point metabolic node that mediates lipid flux through anabolic and catabolic pathways.