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Lipid Mass Spectrometry: Core Technologic Research and Development PAGE

V.) MASS SPECTROMETRY OF LIPID ANTIGENS …………………………………………… 2 A.) Recognition of Lipid Antigens by Natural Killer T Cells ……………………………………… 2 A.1.) Immunologic glycosphingolipidomics and NKT cell development in mouse thymus. ………… 2 A.2.) Invariant natural killer T cells recognize lipid self antigen induced by microbial danger signals………………………………………………………………………………………………. 5 A.3.) Innate and cytokine-driven signals, rather than microbial antigens, dominate in natural killer T cell activation during microbial infection………………………………………………… 6 A.4.) Structural elucidation of diglycosyl diacylglycerol and monoglycosyl diacylglycerol from Streptococcus pneumoniae by multiple-stage linear ion-trap mass spectrometry with electrospray ionization……………………………………………………………………………… 7 A.5.) Recognition of microbial and mammalian phospholipid antigens by NKT cells with diverse TCRs………………………………………………………………………………………... 9 VI.) MASS SPECTROMETRY OF PRODUCTS OF LIPID METABOLIZING ENZYMES INVOLVED IN METABOLIC REGULATION ………………………………….. 12 A.) Fatty Acid Synthase ……………………………………………………………………………... 12 A.1.) New hepatic fat activates PPARα to maintain glucose, lipid, and cholesterol homeostasis…….. 12 A.2.) Identification of a physiologically relevant endogenous ligand for PPARα in liver…………….. 14 A.3.) Macrophage fatty acid synthase deficiency decreases diet-induced atherosclerosis…………….. 18 A.4.) Inhibiting adipose tissue lipogenesis reprograms thermogenesis and PPARγ activation to decrease diet-induced obesity……………………………………………………………………… 21 B.) Group VIA Phospholipase A2 (iPLA2β) ……………………………………………………….. 23 B.1.) Effects of stable suppression of Group VIA phospholipase A2 expression on phospholipid content and composition, insulin secretion, and proliferation of INS-1 insulinoma cells………. 23 B.2.) 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………………………………………………………………………………………… 27 B.3.) Glucose homeostasis, insulin secretion, and islet phospholipids in mice that overexpress iPLA2β in pancreatic β-cells and in iPLA2β-null mice…………………………………………… 29 B.4.) Effects of ER stress on Group VIA PLA2 (iPLA2β) in beta cells include tyrosine phosphorylation and increased association with calnexin……………………………………… 32 B.5.) Group VIA phospholipase A2 (iPLA2β) is activated upstream of p38 MAP kinase in pancreatic islet beta cell signaling……………………………………………………………….. 36 B.6.) Group VIA Phospholipase A2 mitigates palmitate-induced β-cell mitochondrial injury and apoptosis……………………………………………………………………………………… 39 C.) Group VIB Phospholipase A2 (iPLA2γ): Mice deficient in Group VIB Phospholipase A2 exhibit relative resistance to obesity and metabolic abnormalities induced by a Western Diet. …………………………………………………………………………………….. 41 D.) Literature Cited Mass Spectrometry of Lipid Antigens and Products of Lipid Metabolizing Enzymes Involved in Metabolic Regulation …………………………………… 43

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V.) MASS SPECTROMETRY OF LIPID ANTIGENS A.) Recognition of Lipid Antigens by Natural Killer T Cells A.1.) Immunologic glycosphingolipidomics and NKT cell development in mouse thymus. Immunologic glycosphingolipidomics and NKT cell development in mouse thymus. Invariant NKT cells are a hybrid cell type of Natural Killer cells and T cells, whose development is dependent on thymic positive selection mediated by double positive thymocytes through their recognition of natural ligands presented by CD1d, a nonpolymorphic, non-MHC, MHC-like antigen presenting molecule. Genetic evidence suggested that β-glucosylceramide derived glycosphingolipids (GSLs) are natural ligands for NKT cells. N-butyldeoxygalactonojirimycin (NB-DGJ), a drug that specifically inhibits the glucosylceramide synthase, inhibits the endogenous ligands for NKT cells. Furthermore, a β-linked glycosphingolipid, isoglobotriaosylceramide (iGb3), has been found to be a stimulatory NKT ligand. iGb3 synthase knockout mice have a normal NKT development and function, indicating that other ligands exist and remain to be identified. Here a glycosphingolipidomics study of mouse thymus was performed, and mutants were studied that are deficient in β-hexosaminidase b or α-galactosidase A, which are two glycosidases that are up- and downstream agents of iGb3 turnover, respectively. A first database for glycosphingolipids expressed in mouse thymus that are specifically regulated by rate-limiting glycosidases was generated by mass spectrometric analyses. Among identified thymic glycosphingolipids, only iGb3 is a stimulatory ligand for NKT cells, suggesting that large-scale fractionation, enrichment and characterization of minor species of glycosphingolipids are necessary for identifying additional ligands for NKT cells. These results provide insights into cellular lipidomics with a specific focus on glycosphingolipid immunological functions. (J. Proteome Research 2009; 8: 2740–2751).

Figure 1. Glycosphingolipid synthesis in mice. All mouse GSLs stem from β-glucosylceramide or β-galactosylceramide and are assembled by key glycosyltransferaes to become lacto-, ganglio-, globo, isoglobo, sulfo-series complex glycosphingolpids. N-butyldeoxygalactonojirimycin (NB-DGJ) specifically inhibits the synthesis of glucosylceramide derived glycosphingolipids, by inhibiting the glucosylceramide synthase. β4GalT, β1,4 galactosyltransferase 6 (lactosylceramide synthase); CST, cerebroside sulfotransferase.

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Figure 2. Expression of iGb3 and iGb4 in mouse thymus.

Neutral GSLs from wild-type, Hexb KO, and Fabry mice were extracted, permethylated, and analyzed by ion trap mass spectrometry. MS1 ions representing regioisomers of iGb3/Gb3 were selected for MS4 analysis. MS1 ions representing regioisomers of iGb4/Gb4 were selected for MS5 analysis. Percentages of iGb3 in iGb3/Gb3 mixtures were calculated with reference to a standard curve.

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Figure 3. Increased ratio of iGb3 in Gb3 synthase KO mice.

Neutral GSLs from wild type, Gb3 synthase+/- and Gb3 synthase-/- mice were extracted, permethylated, and analyzed by ion trap mass spectrometry. MS1 ions representing regioisomers of iGb3 and Gb3 were selected for MS4 analysis. Percentages of iGb3 in iGb3 and Gb3 mixtures were calculated by reference to a standard curve.

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A.2.) Invariant natural killer T cells recognize lipid self antigen induced by microbial danger signals. Invariant natural killer T cells (iNKT cells) have a prominent role during infection and other inflammatory processes, and these cells can be activated through their T cell antigen receptors by microbial lipid antigens (2). Increasing evidence shows that they are also activated in situations in which foreign lipid antigens would not be present, which suggests a role for lipid self-antigen. We found that an abundant endogenous lipid, β-D-glucopyranosylceramide (β-GlcCer), is a potent iNKT cell self-antigen in mouse and human and that its activity depended on the composition of the N-acyl chain. Furthermore, β-GlcCer accumulates during infection and in response to Toll-like receptor agonists, contributing to iNKT cell activation. Thus, recognition of β-GlcCer by the invariant T cell antigen receptor is proposed to translate innate danger signals into iNKT cell activation (2). (Nat Immunol. 2011; 12: 1202-11)

Figure 4. β-GlcCer is present in primary lymphoid tissues and activates iNKT cells.

(a) TLC analysis of polar lipids from mouse thymus, spleen, whole liver and BMDCs with GSL standards and bovine milk β-GlcCer dose titration. Arrow indicates mobility of β-GlcCer. (b) ELISA of IL-2 production by the iNKT hybridoma DN32 cultured together with RAW cells or CD1d-transfected RAW cells as APCs, with lipid fractions from mouse thymus and spleen, or bovine milk β-GlcCer (fivefold dose titration to a top concentration of 20 µg/ml). (c,d) ESI-MS analysis of β-GalCer purified from thymus (c) and spleen (d), assessed in the electrospray-positive mode and presented relative to the most abundant species, set as 100 (m/z, mass/charge). Major β-GlcCer ions are presented with a lithium adduct; fatty acid composition (determined by collision-induced dissociation tandem mass spectrometry) is in parentheses.. (e) Structures of two abundant β-GlcCer forms detected by ESI-MS.

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A.3.) Innate and cytokine-driven signals, rather than microbial antigens, dominate in natural killer T cell activation during microbial infection. Invariant natural killer T cells (iNKT cells) are critical for host defense against various microbial pathogens, but the central question of how iNKT cells are activated by microbes is not fully understood (3). The example of adaptive MHC-restricted T cells, studies using synthetic pharmacological α-galactosylceramides, and the recent discovery of microbial iNKT cell ligands have all suggested that recognition of foreign lipid antigens is the main driver for iNKT cell activation during infection, but when the role of microbial antigens versus innate cytokine-driven mechanisms were compared, it was found that iNKT cell interferon- γ production after in vitro stimulation or infection with diverse bacteria overwhelmingly depended on toll-like receptor–driven IL-12. Importantly, activation of iNKT cells in vivo during infection with Sphingomonas yanoikuyae or Streptococcus pneumoniae, pathogens which are known to express iNKT cell antigens and which require iNKT cells for effective protection, also predominantly depended on IL-12. Constitutive expression of high levels of IL-12 receptor by iNKT cells enabled instant IL-12–induced STAT4 activation, demonstrating that among T cells, iNKT cells are uniquely equipped for immediate, cytokine-driven activation. These findings reveal that innate and cytokine-driven signals, rather than cognate microbial antigen, dominate in iNKT cell activation during microbial infections (3). (J. Exp. Med. 2011; 208: 1163-77.)

Fig. 5. Detection of microbial lipid antigens expressed by

bacteria.

Lipids were extracted from bacteria and analyzed by ESI-MS. (A–C) [M-H]- adducts of the GSL-1 (GlcAGSL) antigen in Sphingomonas capsulata, N. aromaticvorans, & S. yanoikuyae. (D) [M+CH3COO]- adducts of the GalDAG (BbGL-II) antigen in Bor-rellia burgdorferi. (E) [M+Na]+ adducts of GlcDAG and GalGlcDAG antigens in Streptococcus pneumoniae.

Fig. 6. Innate & cytokine-driven iNKT cell activation during microbial infection. (See Figure on next page). iNKT cell activation during microbial infection is dominantly driven by innate TLR-mediated signals and IL-12, which is released by dendritic cells after stimulation with microbial products. TCR-mediated stimulation also contributes to iNKT cell activation, but the TCR-mediated signal alone, provided either by recognition of CD1d-presented self- or microbial antigens, is not sufficient to result in iNKT cell IFN- γ production in the absence of IL-12. Constitutive expression of high levels of IL-12 receptor endows iNKT cells with ability to respond rapidly to cytokine-mediated stimulation and ensures immediate iNKT cell activation in response to virtually any infectious agent that induces IL-12 production, irrespective of expression of microbial lipid antigens. This allows iNKT cells to overcome their restricted TCR specificity.

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A.4.) Structural elucidation of diglycosyl diacylglycerol and monoglycosyl diacylglycerol from Streptococcus pneumoniae by multiple-stage linear ion-trap mass spectrometry with electrospray ionization. The cell wall of the pathogenic bacterium Streptococcus pneumoniae contains glucopyranosyl diacylglycerol (GlcDAG) and galactoglucopyranosyldiacylglycerol (GalGlcDAG). GlcDAG species with sn-2 vaccenic acid substituents were recently demonstrated to be recognized by invariant natural killer T-cells and thus represent glycolipid antigens. Here, we describe an electrospray ionization (ESI) linear ion-trap (LIT) multiple-stage mass spectrometric (MSn) approach for structural characterization of GalGlcDAG and GlcDAG molecular species (4). MSn (n = 2, 3) of [M+Li]+ adduct ions yields product ions that permit identification of the fatty acid substituents and their positions on the glycerol backbone and location(s) of double bond(s) in the fatty acyl chain. Assignments of the identities and positions of the fatty acid substituents was confirmed by MSn (n = 2, 3) of [M+NH4]

+ ions. The GalGlcDAG and GlcDAG species isolated from S. pneumoniae were found to consist of major species with

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16:1- or 18:1-fatty acid substituents mainly at the sn-2 position with an v-7 (n-7) double bond. More than one isomer was observed for each m/z value in the family. This approach provides a simple method for structural characterization of this important lipid family that would be difficult to achieve with conventional techniques (4). (J. Mass. Spectrom. 2012; 47: 115–123.)

Fig. 7. Proposed fragmentation pathways for structural characterization of 16:0/Δ1118:1-DGDG by CAD of [M+Li]+ (*masses observed for deuterium labeled analogs).

Fig. 8. Electrospray ionization mass spectra of the [M+Li]+ ions of monoglycosyl diacylglycerol (a) and

diglycosyl diacylglycerol (b) isolated from Streptococcus pneumoniae.

A. MONOGLYCOSYL DIAGCYLGLYCEROL

B. DIGLYCOSYL DIAGCYLGLYCEROL

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A.5.) Recognition of microbial and mammalian phospholipid antigens by NKT cells with diverse TCRs. CD1d-restricted natural killer T (NKT) cells include two major subgroups. The most widely studied are Vα14Jα18+ invariant NKT (iNKT) cells that recognize the prototypical α-galactosylceramide antigen, whereas the other major group uses diverse T-cell receptor (TCR) α-and β-chains, does not recognize α-galactosylceramide, and is referred to as diverse NKT (dNKT) cells. dNKT cells play important roles during infection and autoimmunity, but the antigens they recognize remain poorly understood. Here, we identified phosphatidylglycerol (PG), diphosphatidylglycerol (DPG, or cardiolipin), and phosphatidylinositol from Mycobacterium tuberculosis or Corynebacterium glutamicum as microbial antigens that stimulated various dNKT, but not iNKT, hybridomas. dNKT hybridomas showed distinct reactivities for diverse antigens (5). Stimulation of dNKT hybridomas by microbial PG was independent of Toll-like receptor-mediated signaling by antigen-presenting cells and required lipid uptake and/or processing. Furthermore, microbial PG bound to CD1d molecules and plate-bound PG/CD1d complexes stimulated dNKT hybridomas, indicating direct recognition by the dNKT cell TCR. Interestingly, despite structural differences in acyl chain composition between microbial and mammalian PG and DPG, lipids from both sources stimulated dNKT hybridomas, suggesting that presentation of microbial lipids and enhanced availability of stimulatory self-lipids may both contribute to dNKT cell activation during infection (5). (Proc. Nat’l. Acad. Sci. U.S.A. 2013; 110: 1827–1832).

Figure 9. a.) MS2 spectrum of the [M+Li]+ ion m/z 953; (b) MS3 spectrum of m/z 781 (953->791); (c) MS4 spectrum of m/z 611 (953->791->611) (d) MS2 spectrum of the [M+Li]+ ion of m/z 897, (e) and MS4 spectrum of m/z 555 (897->735->555). Ions marked with ‘O’ locate double bonds along the 18:1-fatty acid (FA) (c) and 16:1-FA (e) substituents, respectively.

(See Figure on Next Page). Figure 10. Isolation of stimulatory Mtb lipids. (A) Using DEAE cellulose columns, Mtb polar lipids were separated into 10 semi-preps (SP) that were tested for stimulation of 14S.10 cells. (B) Lipids from fraction 4.3 were further separated by preparative 1D & 2D TLC, resulting in the stimulatory 4.3.2 Mtb lipid. (C & D) MS analyses revealed lipid 4.3.2 (C) to be various molecular species of diradyl-glycerophosphoglycerol (GPG) lipids (D).

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Fig. 11. Antigen specificities of Vα14+ iNKT, Vα10+ NKT, and dNKT cells. GSL-1 is found in Sphingomonas spp., Bb DAG in Borrelia burgdorferi, and Ms α-GlcA DAG in Mycobacterium smegmatis.

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Figure 12. Cg lipids stimulate dNKT hybridomas. (A) Polar or apolar Cg lipids were tested for reactivity of dNKT hybridomas as in Fig. 1B. (B) Cg polar lipids were separated by 2D TLC and analyzed for reactivity with 14S.10 cells. (C–E) MS analysis revealed (C) the stimulatory lipid p6 to be 18:1/16:0-GPG (C) and the active lipid p5 (D) to be cardiolipin (CL) (E). (F) Stimulation of 14S.10 cells in the presence of RAW.CD1d or RAW.ut cells and purified Mtb or Cg GPG.

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VI.) MASS SPECTROMETRY OF PRODUCTS OF LIPID METABOLIZING ENZYMES INVOLVED IN METABOLIC REGULATION

A.) Fatty Acid Synthase

A.1.) New hepatic fat activates PPARα to maintain glucose, lipid, and cholesterol homeostasis. De novo lipogenesis is an energy-expensive process whose role in adult mammals is poorly understood. Here, mice were generated with liver-specific inactivation of fatty-acid synthase (FAS), a key lipogenic enzyme. On a zero-fat diet, FASKOL (FAS knockout in liver) mice developed hypoglycemia and fatty liver, which were reversed with dietary fat (6). These phenotypes were also observed after prolonged fasting, similarly to fasted PPARα-deficiency mice. Hypoglycemia, fatty liver, and defects in expression of PPARα target genes in FASKOL mice

were corrected with a PPARα agonist. On either zero-fat or chow diet, FASKOL mice had low serum and hepatic cholesterol levels with elevated SREBP-2, decreased HMG-CoA reductase expression, and decreased cholesterol biosynthesis; these were also corrected with a PPARα agonist. These results suggest that products of the FAS reaction regulate glucose, lipid, and cholesterol metabolism by serving as endogenous activators of distinct physiological pools of PPARα in adult liver (6). (Cell Metabolism 2005; 1: 309-322).

Fig. 13. Targeting the fatty-acid synthase gene. A) Wild-type, targeting vector, and fatty-acid synthase (FAS) lox allele before & after recombination. Post-Cre allele depicts deletion of exons 4–8 (gray shaded boxes). NEO, neomycin positive-selection cassette; open boxes,

exons preserved; black arrowheads, loxP sites; open arrowheads, genotyping primers. B) Southern blot DNA analyses of control (W, lox−/− Cre+; F−, lox+/+ Cre−) and FASKOL (F, lox+/+ Cre+) mice. Genomic DNA (from liver, lung, heart, brain, kidney, and skeletal muscle) was digested with NdeI

and HindIII and detected with probe C to yield expected 7.3 kb (allele without loxP), 8.9 kb (floxed allele), and 5.3 kb (post-Cre allele) fragments. C) PCR analyses. DNA from FASKOL livers (lox+/+ Cre+) produced a 317 bp product (lane 3) indicating appropriate deletion at the FAS gene. The product was absent from livers of wt mice of various genotypes and from hearts of FASKOL mice (lane 7). D) Assessing FAS activity (upper panel) and malonyl-CoA content (lower panel). Liver homogenates from overnight-fasted 12 hr chow-refed male FASKOL (black bar) and littermate control mice of various genotypes aged 16–20 weeks were assayed for FAS activity and malonyl-CoA content. Inset of (D) represents FAS activity in lung of FASKOL (black bar) and wt (lox+/+ Cre−, white bar) mice. E) GC-MS analyses of hepatic palmitate (C16:0) and palmitoleate (C16:1) from chow and zero-fat diet (ZFD) fed wt (lox+/+ Cre−) and FASKOL mice. Peak identities were verified by full EI-MS scans.

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FIG. 14. Effects of PPARα activation on glucose and lipid homeostasis in FASKOL and wt (lox+/+ Cre−) mice A–J). Measurements of serum glucose (A), insulin (B), glucagon (C), insulin to glucagon ratio (D), liver glycogen (E), liver triglycerides (F), serum triglycerides (G), serum nonesterified fatty acids (H), serum total cholesterol (I), and liver cholesterol content (J) were obtained from 4 hr fasted wt and FASKOL mice on chow or zero-fat diet for 10 d (ZFD), or PPARα agonist Wy14,643 added to chow (C+Wy) or ZFD (Z+Wy) for 10 d. p < 0.05 for the following: *, compared to the corresponding wt mice; #, compared to the corresponding chow-fed mice; **, compared to the corresponding ZFD-fed mice.

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A.2.) Identification of a physiologically relevant endogenous ligand for PPARα in liver. The nuclear receptor PPARα is activated by drugs to treat human disorders of lipid metabolism. Its endogenous ligand is unknown. PPARα-dependent gene expression is impaired with inactivation of fatty acid synthase (FAS), suggesting that FAS is involved in generation of a PPARα ligand. Here the FAS-dependent presence of a phospholipid bound to PPARα isolated from mouse liver is demonstrated (7). Binding was increased under conditions that induce FAS activity and displaced by systemic injection of a PPARα agonist. Mass spectrometry identified the species as 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (16:0/18:1-GPC). Knockdown of the enzyme Cept1, which is required for phosphatidylcholine synthesis, suppressed PPARα-dependent gene expression. Interaction of 16:0/18:1-GPC with the PPARα ligand-binding domain and coactivator peptide motifs was comparable to PPARα agonists, but interactions with PPARγ were weak and none were detected with PPARγ. Portal vein infusion of 16:0/18:1-GPC induced PPARα-dependent gene expression and decreased hepatic steatosis. These data suggest that 16:0/18:1-GPC is a physiologically relevant endogenous PPARα ligand (7). (Cell 2009; 138: 476–488).

Figure 15. Model for differential effects of hepatic lipid. Fat absorbed from the diet or synthesized de novo via FAS in the liver constitutes “new” fat, capable of activating PPARα to ensure normal glucose and lipid homeostasis. Fat derived from peripheral mobilization of adipose stores constitutes a different hepatic compartment (“old” fat) that does not appear to activate PPARα as effectively as new fat, leading to fatty liver. In contrast to de novo synthesized fat, dietary fat is inadequate for the maintenance of cholesterol homeostasis, suggesting different PPARα pools.

(See Figure on Next Page). Figure 16. Generation of Liver-Specific FAS Knockout Mice on a PPARα -Null Background and Reconstitution of Liver PPARα Expression. (A) PCR analysis. Liver DNA was amplified using primer sets for the FAS floxed allele (top), PPARα (middle), and Cre (bottom). (B) Immunoblot analysis of liver lysates for wild-type (WT) and FASKOL mice on a PPARα null background via FAS (top) and actin (bottom) antibodies. (C and D) FAS activity (C) and malonyl-CoA content (D). Liver homogenates from overnight-fasted 12 hr chow-refed male WT and FASKOL mice on a PPARα

null background were assayed. *p < 0.05. (E) Diagram for isolation of FLAG-tagged PPARα. (F) Immunoprecipitation (IP) and immunoblot (IB) analysis in livers of WT and FASKOL mice on a PPARα null background infected with adenoviruses encoding GFP alone (AdGFP) or FLAG-tagged PPARα (AdFLAG-PPARα). Nuclear fractions were immunoprecipitated with FLAG antibodies and immunoblotted with either FLAG antibody (top) or PPARα antibody (middle). Crude liver lysates were immunoblotted with actin antibody (bottom).

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Figure 17. Identification of a Glycerophosphocholine Species in FLAG-Eluted Hepatic Nuclear Extracts.

Positive ion ESI/MS analyses of lithiated adducts of hepatic nuclear phospholipids were performed to monitor neutral loss of 189 [LiPO4(CH2)2N(CH3)3], which identifies parent [MLi+] ions that contain the phosphocholine head-group in lipid mixtures. (A–D) Representative profiles of glycerophosphocholine (GPC) species in chow-fed WT and FASKOL mice on a PPARα null background infected with AdGFP (A and C) or AdFLAG-PPARa (B and D). (E–H) Representative profiles of GPC species in zero-fat diet (ZFD)-fed WT and FASKOL mice on a PPARα null background infected with AdGFP (E and G) or AdFLAG-PPARα (F and H). Insets in (B), (D), (F), and (H) depict the fragment ion at mass-to-charge ratio (m/z) 766 as the specific GPC species that is both PPARα and FAS dependent. (I) Quantification of the relative abundance of the m/z 766 ion with respect to genotype (W/P, WT on PPARα null background; F/P, FASKOL on PPARα null background) and diet (chow and ZFD). Each bar represents mean ± SEM from 3 independent experiments with 4–6 mice in each group per experiment. *p < 0.05 versus corresponding W/P control; **p < 0.05.

Figure 18. Tandem MS Identifies the GPC Species as 1-Palmitoyl-2- Oleoyl-sn-Glycerol-3-

Phosphocholine

(A) Fragmentation pattern upon collisionally activated dissociation of the ion of m/z 766, which corresponds to the lithiated adduct [MLi+] of 16:0/18:1-GPC. (B) Expansion of the mass spectrum in (A) from m/z 400 to m/z 540 to illustrate relative abundances of ions that represent losses of fatty acid substitutents. The data indicate that palmitate and oleate are the sn-1 and sn-2 substituents, respectively. (C) Structure of the putative PPARα ligand.

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Figure 19. In vivo displacement of the endogenous PPARα ligand with a PPARα agonist. Li+ adducts of GPC species in excess FLAG-eluted hepatic nuclear extracts obtained from Wy14,643 (Wy)-treated mice were analyzed by positive ion ESI/MS/MS scans for neutral loss of 189, reflecting elimination of lithiated phosphocholine from [MLi+]. (A–D) ESI/MS/MS scans of GPC species at baseline (time 0) (A), 10 min (B), 30 min (C), and 60 min (D) after an intraperitoneal injection of 50 mg/g Wy14,643 in chow-fed WT mice on a PPARα null background injected with AdFLAG-PPARα adenovirus. (E–H) ESI/MS/MS scans of GPC species at baseline (time 0) (E), 10 min (F), 30 min (G), and 60 min (H) after the same treatment in ZFD-fed mice. Insets in (A)–(H) depict the ion m/z 766 (16:0/18:1-GPC) that is displaced in a time-dependent manner by Wy14,643. (I) Quantitation of relative abundance of m/z 766 ion in response to Wy14,643.

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Figure 20. Portal vein infusion of 16:0/18:1-GPC rescues hepatic steatosis in a PPARα -dependent manner.

(A) Operative field depicting the portal vein (PV) cannulated with a catheter (pv-cath) positioned at the entry site into the liver (lvr). The catheter is intentionally marked in black ink at its proximal tip to enhance visualization. Labels indicate gall bladder (gb), bile duct (bd), inferior vena cava (ivc), and pancreas (pan). (B) Intraportal 16:0/18:1-GPC treatment protocol. After insertion of the portal vein catheter, C57BL/6 mice (wild-type for FAS and either wild-type or null for PPARα) were allowed to recover. On day 4, chow was changed to a zero-fat diet (ZFD) and mice received 3 intraportal injections/day of 10 mg/kg 16:0/18:1-GPC sonicated in normal saline/0.5% ethanol/0.5% fatty acid-free BSA or vehicle alone. On the last day before the end of treatment (day 9), mice were fasted for 24 hr. (C) Liver sections were stained with oil red O at the end of treatment to visualize neutral lipids from wild-type (C57/BL6) and PPARα-/- mice treated with 16:0/18:1-GPC or vehicle (Veh). (D) Quantification of hepatic triglyceride content per unit mass of tissue from vehicle and 16:0/18:1-GPC treated C57/BL6 and PPARα-/- mice. *p < 0.05 vs. corresponding Veh. #p < 0.05 vs. C57/BL6 controls. (E) Expression of hepatic Acox1 (ACO; top) and Cpt1a (CPT-1; bottom) mRNA by RT-PCR normalized to control L32 ribosomal mRNA after the 16:0/18:1-GPC injections.. *p < 0.05 vs. corresponding Veh. #p < 0.05 vs. C57/BL6 controls. (F) Proposed model for generation of the endogenous PPARα ligand in liver. FAS yields palmitate (C16:0), and 16:0/18:1-GPC is likely generated through the diacylglycerol (DAG) intermediate and the enzymatic activity of CEPT1 either in the ER or the nucleus. Binding of 16:0/18:1-GPC to PPARα in the nucleus activates transcription machinery (TM) turning on PPARα-dependent genes and affecting hepatic lipid metabolism. ACC, acetyl CoA carboxylase; ER, endoplasmic reticulum.

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A.3.) Macrophage fatty acid synthase deficiency decreases diet-induced atherosclerosis. Fatty acid metabolism is perturbed in atherosclerotic lesions, but whether it affects lesion formation is unknown. Here fatty-acid synthase (FAS) was inactivated in macrophages of apoE-deficient mice to determine whether fatty acid synthesis affects atherosclerosis. Serum lipids, body weight, and glucose metabolism were the same in FAS knock-out in macrophages (FASKOM) and control mice, but blood pressure was lower in FASKOM animals (8). Atherosclerotic extent was decreased 20–40% in different aortic regions of FASKOM as compared with control mice on Western diets. Foam cell formation was diminished in FASKOM as compared with wild type macrophages due to increased apoAI-specific cholesterol efflux and decreased uptake of oxidized low density lipoprotein. Expression of the anti-atherogenic nuclear receptor liver X receptor-α (LXRα; Nr1h3) and its downstream targets, including Abca1, were increased in FASKOM macrophages, whereas expression of the potentially pro-atherogenic type B scavenger receptor CD36 was decreased. Peroxisome proliferator-activated receptor-α (PPARα) target gene expression was decreased in FASKOM macrophages. PPARα agonist treatment of FASKOM and wild type macrophages normalized PPARα target gene expression as well as Nr1h3 (LXRα). Atherosclerotic lesions were more extensive when apoE null mice were transplanted with LXRα-deficient/FAS-deficient bone marrow as compared with LXRα-replete/FAS-deficient marrow, consistent with antiatherogenic effects of LXRα in the context of FAS deficiency. These results show that macrophage FAS deficiency decreases atherosclerosis through induction of LXRα and suggest that FAS, which is induced by LXRα, may generate regulatory lipids that cause feedback inhibition of LXRα in macrophages (8). (J. Biol. Chem. 2010; 285: 23398–23409).

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Figure 21. Targeting FAS in macrophages. A, top shows the targeted allele (FAS locus with loxP-flanked exons 4–8 (with the loxP sites indicated as triangles)) and the post-Cre allele. P1 and P2 represent primers used to detect a 317-bp fragment indicating Cre excision at the FAS locus. The bottom shows a simplified breeding scheme (omitting intermediate progeny) to produce FASKOM mice. B, PCR analyses of DNA from FASKOM and control mice using P1 and P2. DNA sources for both FASKOM and WT were as follows: lane 1, liver; lane 2, spleen; lane 3, lung; lane 4, kidney; lane 5, heart; lane 6, aorta; lane 7, adipose tissue; lane 8, peritoneal macrophages; and lane 9, negative control. C, FAS mRNA expression in peritoneal macrophages by quantitative RT-PCR. Data are expressed relative to L32 mRNA. *, p<0.05. D, FAS enzyme activity in peritoneal macrophages. *, p<0.001. The inset shows FAS enzyme activity in liver.

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(See Figure on Preceding Page) Figure 22. Cholesterol metabolism in peritoneal macrophages. A, Oil Red O staining of peritoneal macrophages (MØ). B, Lipid content of peritoneal MØ. *, p < 0.05. C, Electrospray ionization-mass spectrometric analysis of fatty acids from WT and FASKOM peritoneal MØ. The y-axis represents relative abundance and the x-axis m/z values. The m/z 311.3 peak is an internal standard. D, ApoAI-specific cholesterol efflux from MØ of each genotype. Cells were loaded with acetylated LDL in the presence of [3H]cholesterol and then treated with apoAI for 48 h. *, p<0.05. E, Western blot of proteins extracted from WT and FASKOM MØ and probed with an anti-ABCA1 antibody (top panel) and an anti-actin antibody (bottom panel). F, cholesterol uptake at 4 and 16 h after exposure to fluorescently labeled oxidized LDL using MØ of each genotype. *, p<0.05.

Figure 23. Lipid content in atherosclerotic lesions. WT and FASKOM mice were fed a high fat diet for 8 wk; mice were visually selected for differences in atherosclerosis and then the aortic arch was isolated without fixation, cleaned, and subjected to lipid extraction. For these mice, serum cholesterol at the time of sacrifice was 1747±217 mg/dl for WT and 1842±170 mg/dl for FASKO. A, cholesterol content in the aortic arch for each genotype. *, p<0.05. B, total lipid phosphorus in aortic arches. C, total triglyceride content in aortic arches. D, ESI/MS analysis of triacylglycerol species extracted from aortic arches of WT and FASKOM mice. The x-axis represents m/z values and the y-axis relative abundance in arbitrary units. The internal standard is not shown to simplify data presentation.

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A.4.) Inhibiting adipose tissue lipogenesis reprograms thermogenesis and PPARγ activation to decrease diet-induced obesity. De novo lipogenesis in adipocytes, especially with high fat feeding, is poorly understood. Here, an adipocyte lipogenic pathway encompassing fatty acid synthase (FAS) and PexRAP (peroxisomal

reductase activating PPARγ) is demonstrated to modulate endogenous PPARγ activation and adiposity (9). Mice lacking FAS in adult adipose tissue manifested increased energy expenditure, increased brown fatlike adipocytes in subcutaneous adipose tissue, and resistance to diet-induced obesity. FAS knockdown in embryonic fibroblasts decreased PPARγ transcriptional activity and adipogenesis. FAS-dependent alkyl ether phosphatidylcholine species were associated with PPARγ and treatment of 3T3-L1 cells with one such ether lipid increased PPARγ transcriptional activity. PexRAP, a protein required for alkyl ether lipid synthesis, was associated with peroxisomes and induced during adipogenesis. PexRAP knockdown in cells decreased PPARγ transcriptional activity and adipogenesis. PexRAP-knockdown in mice decreased expression of PPARγ dependent genes and reduced diet-induced adiposity. These findings suggest that inhibiting PexRAP or related lipogenic enzymes could treat obesity and diabetes (9). (Cell Metab. 2012; 16: 189-201.)

Figure 24. Isolation of FAS-dependent diacyl & 1-O-alkyl ether phosphatidylcholine species associated with PPARγ . (A) Strategy for detection of PPARγ-associated lipids. (B) Detection of FLAG-PPARγ protein immunoprecipitated from adipocytes treated with control or FAS shRNA. (C) Mass spectrometric analyses of [M+Li]+ ions of glycerophosphocholine (GPC) lipids bound to FLAG-PPARg or control protein (GFP) immunoprecipitated from control and FAS knockdown adipocytes. Ions of m/z 752 and 780 represent 1-O-alkyl GPC species. (D) CV-1 cells were transfected with plasmids encoding UAS-luciferase, Renilla luciferase and Gal4-PPARγ LBD (a C-terminal fragment of PPARγ encompassing the ligand binding domain fused to the Gal4 DNA binding domain) or Gal4 alone. Cells were treated with 18:1e/16:0-GPC (corresponding to m/z 752 in C), rosiglitazone, or DMSO. After 48 hr, UAS-luciferase reporter activity was measured and normalized to Renilla luciferase reporter activity. **p = 0.0001. *p = 0.018 (10 mM), 0.024 (20 mM), 0.019 (80 mM). (E) 3T3-L1 cells were induced to differentiate in DMEM+10% FBS with supplemental dexamethasone, insulin and IBMX in the presence of 20 mM 18:1e/16:0-GPC or DMSO. After 3 d, cells were retreated with GPC in media containing supplemental insulin alone. The next day, cells were harvested for RNA extraction and real-time PCR. *p = 0.0147 (aP2), 0.0006 (LPL), 0.0102 (CD36).

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Figure 25. Cloning and characterization of the terminal component in the mammalian peroxisomal ether lipid synthetic pathway.

(A) The peroxisomal acyl-DHAP pathway of lipid synthesis. FAS, fatty acid synthase; ACS, acyl CoA synthase; G3PDH, glycerol 3-phosphate dehydrogenase; DHAP, dihydroxyacetone phosphate; DHAPAT, DHAP acyltransferase; FAR1, fatty acyl CoA reductase 1; ADHAPS, alkyl DHAP synthase; ADHAP Reductase, acyl/alkyl DHAP reductase activity; LPA, lysophosphatidic acid; AGP, 1-O-alkyl glycerol 3-phosphate. (B) Mouse DHRS7b is homologous to yeast acyl DHAP reductase, Ayr1p. TMD, transmembrane domain; Adh_short, short chain dehydrogenase/reductase domain. (C) PexRAP (peroxisomal reductase activating PPARγ, detected using anti-DHRS7b antibody) is enriched in peroxisomal fractions isolated from 3T3-L1 adipocytes. S, supernatant; P, pellet after sedimentation. (D) Pex19 coimmunoprecipitates with Myc-tagged PexRAP. WCL, whole cell lysates. (E) Pex19 interacts with PexRAP in GST pull-down experiments using 3T3-L1 adipocytes. (F) RT-PCR analysis of PexRAP expression with PexRAP knockdown in MEFs. **p = 0.0084. (G) Mass spectrometric analyses of [M+H]+ ions of GPC lipids in MEFs after PexRAP knockdown. Quantification of the 1-O-alkyl ether GPC lipid peak at m/z 746 [M+H]+ (identical to the lithium adduct at m/z 752) is shown in the inset. **p = 0.0009. (H) Mouse tissue distribution of PexRAP protein by western blotting. (I) Protein abundances of PexRAP and FAS increase prior to increases in C/EBPa and aP2 during differentiation of 3T3-L1 adipocytes.

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B.) Group VIA Phospholipase A2 (iPLA2β)

B.1.) 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 (10). 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

Figure 26. Knockdown of PexRAP in mice alters body Composition and metabolism.

(A) PexRAP knockdown using antisense oligonucleotides (ASOs) in Hepa1-6 cells. (B) Western blot analysis using epididymal WAT of C57BL/6J mice treated intraperitoneally with the indicated doses of control or PexRAP ASO twice a week for 3 weeks. (C) RT-PCR analysis of epididymal WAT expression following ASO treatment. P vs. control: PexRAP **0.0078; PPARg **0.0051; CD36 *0.0420; LPL **0.0030. (D) Body composition by MRI following 4 wk HFD feeding (baseline) and after 3.5 wk ASO treatment while still eating HFD. **p = 0.0098 for fat, 0.0071 for lean. (E) Glucose tolerance testing in HFD-fed mice following ASO treatment. *p = 0.0220 at 15 min and 0.0434 at 120 min. **p = 0.0019. (F) Insulin levels at 30 min point from (E). *p = 0.0363. (G) Models of PPARγ and PPARα gene expression in WT and FAS-deficient adipocytes.

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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 (10). This calls into question the generality

Fig. 27. Suppression of iPLA2β expression in iPLA2β knockdown INS-1 cells. INS-1 cell lines were prepared with retroviral vectors containing an insert encoding scrambled RNA (control) or siRNA against iPLA2β mRNA to generate iPLA2β knockdown cell lines, and iPLA2β mRNA was analyzed by Northern blots (A, lane 1, vector control; lane 2, iPLA2β-KD1; lane 3, iPLA2β-KD2; lane 4, parental cells) and real time PCR (B). Activity of iPLA2β (C) was measured without Ca2+ in the presence of EGTA and without (open bars) or with 1 mM ATP alone (cross-hatched bars) or with ATP and 10 µM BEL (solid bars). The leftmost bar (B) or set of bars (C) reflects control cells, and the center and rightmost bar or set of bars reflects iPLA2β-KD1 and iPLA2β-KD2 cells, respectively.

Fig. 28. Effects of glucose and the adenylyl cyclase activator forskolin on insulin secretion from iPLA2β knockdown and control INS-1 cells. Insulin secretion by control, iPLA2β-KD1, and iPLA2β-KD2 INS-1 cells was measured after a 1-h incubation in medium containing 3 or 20mM glucose without or with 2.5 µM forskolin. Mean values ± S.E. (n = 6) normalized to cell protein content are displayed. Values for iPLA2β-KD INS-1 cells were significantly lower (p < 0.05) than control under all conditions.

Fig. 29. Rates of proliferation of control and iPLA2β knockdown INS-1 cells. INS-1 cells (0.3 x 106 cells/well) were cultured (37°C) for various intervals and then detached with trypsin/EGTA solution, and cell number was determined from fluorescence enhancement upon association of CyQuant indicator withDNA (A) or from BrdUrd incorporation (B). Mean values ± S.E. are indicated (n = 6). Values for iPLA2β-KD1 and iPLA2β-KD2 cells were significantly lower (p < 0.05) than control at 1 and 3 days.

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of proposed housekeeping functions for iPLA2β in PC homeostasis and remodeling. J. Biol. Chem. 2006; 281: 187–198.)

(See lower left figure on preceding page). Fig. 30. Electrospray ionization mass spectrometric analyses of INS-1 cell glycerophosphocholine lipids. Phospholipids from control (A) or iPLA2β-KD1 INS-1 cells (B) were analyzed as Li+ adducts by positive ion ESI/MS. In C and D, iPLA2β-KD1 INS-1 cells were incubated for 24 h with 10 or 30 µM supplemental arachidonic acid, respectively, in the culture medium before lipids were extracted and analyzed.

(See lower right figure on preceding page). Fig. 31. Tandem MS of arachidonate-containing glycerophosphocholine lipids in INS-1 cells cultured with arachidonic acid (AA). GPC lipid-Li+ adducts from control or iPLA2β-KD1 INS-1 cells incubated with AA were analyzed by positive ion ESI/MS/MS. A & B, CAD of m/z 788, and product ions were analyzed. A, ions from m/z 50-800; B, ions from m/z 300-620. In C & D, CAD of m/z 816.

Figure 32. Electrospray ionization mass spectrometric analyses of glycerophosphocholine lipids in iPLA2β knockdown and control INS-1 cells incubated with arachidonic acid for various intervals. Control (A–C) or iPLA2β-KD (D–F) INS-1 cells were incubated with arachidonic acid for 0 h (A and D), 6 h (B and E), or 24 h (C and F). Extracted GPC lipid-Li+ adducts were analyzed by positive ion ESI/MS/MS scanning for neutral loss of 183.

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FIG. 33. ESI/MS analyses of INS-1 cell lysophosphatidylcholine species. Extracted lipids from control (A) or iPLA2β-KD1 INS-1 cells (B) at time 0 or after 24 h (D) of incubation with arachidonic acid were analyzed as Li+ adducts by positive ion ESI/MS/MS for neutral loss of 59. In C, the ion m/z 528 from ESI/MS analyses in A or B was subjected to CAD, and product ions were analyzed. D, the ESI/MS/MS constant neutral loss of 59 scan for LPC from INS-1 cells incubated (24 h) with arachidonic acid.

Fig. 34. Tandem MS of arachidonate-containing lysophos-phatidylcholine species from INS-1 cells cultured with arachi-donic acid and standard (1-arachidonoyl, 2-lyso)-sn-glycero-phosphocholine. Extracted lipids from control or iPLA2β-KD1 INS-1 cells incubated for 24 h with arachidonic acid were analyzed as Li+ adducts by positive ion ESI/MS/MS, the ion m/z 550 was subjected to CAD, and its product ions were analyzed. A, the spectrum obtained with fresh extracts; B, the spectrum obtained after the extract had been stored for 2 weeks. C, the tandem spectrum of standard 1-arachidonoyl, 2-lyso-GPC prepared from 20:4/20:4-GPC with N. naja phospholipase A2.

Fig. 35. Negative ion ESI/MS analyses of anionic glycerophospholipids in iPLA2β-KD and control INS-1 cells incubated with arachidonic acid. Lipid phosphorus was measured in extracts of iPLA2β-KD (A & B) or control (C & D) INS-1 cells incubated with arachidonic acid for 0 h (A and C) or 24 h (B and D); internal standard 14:0/14:0-GPE was added; and the mixture was analyzed by negative ion ESI/MS.

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B.2.) 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 (11). 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

Fig. 36. Expression of iPLA2β mRNA in tissues of wild-type and iPLA2β-null mice. A represents Northern blot analyses of total RNA from wild-type (WT or W) and iPLA2β-null (KO or K) mouse brain, kidney, skeletal, muscle, aorta, liver, and isolated pancreatic islets. The upper panels were probed for iPLA2β mRNA, and the lower panels were probed for 18 S ribosomal RNA. B depicts analyses of products from RT-PCR amplification of iPLA2β mRNA fragments using total RNA isolated from pancreatic islets and brain as template and two different sets of primers. Lane M represents molecular weight standards. Lanes 1, 4, and 7 represent WT tissue, and lanes 2, 5, and 8 represent iPLA2β-knock-out (KO) tissues. Lanes 3, 6, 9, and 10 represent control reactions in which no template was added. In lanes 1–3, a primer set was used that amplifies an mRNA fragment that spans the site of insertion of the neomycin resistance cassette in the knock-out construct. In lanes 4–6, a primer set was used that amplifies a fragment downstream from the neomycin resistance cassette insertion site. In lanes 7–9, a primer set was used to amplify a fragment of actin mRNA as control.

Fig. 37. Fasting blood glucose and insulin concentrations as a function of time after administration of multiple low doses of streptozotocin to wild-type or iPLA2β-null mice (Upper Panels). Wild-type (WT, closed squares) or iPLA2β-null (KO, closed circles) mice were treated with intraperitoneal (IP) streptozotocin (STZ) injections and then fed normal chow. Fasting blood glucose (A) and insulin (B) concentrations were determined after STZ injection. Glucose values are expressed as % of the time 0 value. Glucose tolerance testing (GTT) of WT and iPLA2β-KO mice fed normal chow (NC) or a high fat diet (HFD) (Lower Panels). WT (A) or KO (B) mice were fed NC (closed squares) or HFD (closed circles) for 6 mo. IP GTT was performed after an overnight fast.

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fat diet (11). These and previous findings thus indicate that iPLA2β-null mice exhibit phenotypic abnormalities in pancreatic islets in addition to testes and macrophages. (J. Biol. Chem. 2006: 281: 20958-20973.)

Fig. 38. ESI/MSc analyses of glycerophosphocholine lipids from brain and pancreatic islets of wild-type and iPLA2β-null mice. Phospholipids from wild-type (A and C) or iPLA2β-null (B and D) mouse brain (A and C), or pancreatic islets (B and D) were analyzed as Li+ adducts by positive ion ESI/MS, and the relative abundances of the ion currents were plotted as a function of m/z value.

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Fig. 39. Tandem MS of glycerophosphocholine lipids from mouse brain and islets. GPC lipid-Li+ adducts from WT or iPLA2β

-/- mouse brain extracts were analyzed by positive ion ESI/MS/MS. Ions selected in the first mass-analyzing quadrupole were accelerated into the collision cell to induce CAD. Product ions were analyzed in the final quadrupole. Ions selected for CAD included m/z 788 (A), 816 (B), 766 (C ), &794 (D).

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B.3.) Glucose homeostasis, insulin secretion, and islet phospholipids in mice that overexpress iPLA2β in pancreatic β-cells and in iPLA2β-null mice. 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-elevatingagents. 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 iPLA2 overexpression in RIPiPLA2β-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. (Am. J. Physiol. Endocrinol. Metab. 2008; 294: E217–E229.)

Fig. 40. Insulin tolerance testing (ITT) of wild-type (WT) and iPLA2β-null mice that had been fed normal chow or a high fat diet. Wild type (closed squares) or iPLA2β-null (closed circles) mice were fed normal chow after weaning until 8 weeks of age and thereafter were fed a high fat diet for 6 months. Intraperitoneal insulin tolerance testing was then performed after an overnight fast.

Fig. 41. Insulin secretion from pancreatic islets isolated from wild type (WT) and iPLA2β-null mice that had been fed a high fat diet. Pancreatic islets were isolated from WT (black bars) or iPLA2β-null (cross-hatched bars) mice that had been fed a high fat diet and incubated at 3, 8, or 20 mM glucose in the absence or presence of 2.5 µM forskolin. Medium was then removed for measurement of insulin by radioimmunoassay.

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Figure 42. Overexpression of iPLA2β in pancreatic islets of RIP-iPLA2β-TG mice.

A: real time PCR analyses of iPLA2β mRNA levels in islets from wild-type (WT) or RIPiPLA2β-TG mice of line TG1 or TG2. B: Western blot of immunoreactive iPLA2β protein in pancreatic islets isolated from WT or RIP-iPLA2β-TG mice of line TG1 or TG2. Immunoblots were also probed with actin antibodies as a loading control. C: iPLA2β-specific enzymatic activity measurements in islets from WT or RIP-iPLA2β-TG mice from line TG1. Black bars represent basal specific activity, and light gray bars represent activity in presence of ATP. Third (rightmost) bar in each set represents activity in presence of iPLA2 suicide substrate (E)-6-(bromomethylene) tetrahydro-3-(1-naphthalenyl)-2Hpyran-2-one (BEL). Mean values SE (n = 6) are displayed in A and C. *P<0.05 for WT vs. RIP-iPLA2β-TG.

Fig. 43. Intraperitoneal glucose and insulin tolerance tests with RIP-iPLA2β-TG and WT male mice. In A, D-glucose (2 mg/g) and in B human regular insulin (0.75 U/kg) was administered by intraperitoneal injection to WT (■) or RIP-iPLA2β-TG () male mice 20–24 wk of age, and blood was collected at baseline and at 30, 60, and 120 min after injection to measure glucose concentration.. *P 0.05 for WT vs. TG.

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Figure 44. Upper Panels: Electrospray ionization tandem mass spectrometric analyses (ESI/MS/MS) of diradyl-GPC lipids from pancreatic islets of WT and RIP-iPLA2β-TG mice. Phospholipids from pancreatic islets of WT (A) & RIPiPLA2β-TG (B) mice were mixed with internal standard 14:0/14:0-GPC and were analyzed as Li+ adducts by positive ion ESI/MS/MS scanning for neutral loss of 183 (phosphocholine), and relative abundances of ion currents were plotted vs. m/z value. Lower Panels: ESI/MS/MS of the lysophosphatidylcholine (LPC) content of islets of WT & RIP-iPLA2β-TG mice. Phospholipids were extracted from islets of WT (A) & RIP-iPLA2β-TG (B) mice, mixed with internal standard 19:0-LPC, and analyzed as Li+ adducts by positive ion ESI/MS/MS scanning for neutral loss of 59 to visualize LPC species.

Figure 45. RIP-iPLA2β-TG mouse islets have increased glucose-induced action potential duration with decreased frequency and corresponding changes in glucose-induced elevation of cytosolic [Ca2+] vs. WT islets. Electrical activity induced by 20 mM glucose is recorded for WT control (A) or RIP-iPLA2β-TG (B) mouse β-cells. Insets display action potentials (APs) from segment of activity indicated by horizontal bars. Lowest tracings in each panel represent fast-acquisition [Ca2+] traces recorded from same entire mouse islet during segment of activity indicated by horizontal bars, loaded with fluo 4, and imaged at a frequency of 10 kHz. Decrease in AP frequency of WT control vs. RIP-iPLA2β-TG mouse islets (from 1.7/s to 0.96/s) taken at 5 min after glucose was significant(P< 0.035).

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B.4.) Effects of ER stress on Group VIA PLA2 (iPLA2β) in beta cells include tyrosine phosphorylation and increased association with calnexin. The Group VIA phospholipase A2 (iPLA2β) hydrolyzes glycerophospholipids at the sn-2-position to yield a free fatty acid and a 2-lysophospholipid, and iPLA2β has been reported to participate in apoptosis, phospholipid remodeling, insulin secretion, transcriptional regulation, and other processes. Induction of endoplasmic reticulum (ER) stress in β-cells and vascular myocytes with SERCA inhibitors activates iPLA2β, resulting in hydrolysis of arachidonic acid from membrane phospholipids, by a mechanism that is not well understood. Regulatory proteins interact with iPLA2β, including the

Figure 46. Delayed rectifier currents in pancreatic islet β-cells from WT control and RIP-iPLA2β-TG mice. Kv current traces recorded from islet β-cells from WT control (A) or RIP-iPLA2β-TG mice (B) incubated in medium without glucose (left tracings) or with 20 mM glucose (right tracings) and subjected to 500-ms depolarization in 10 mV and 5 mV increments from 80 mV to 30 mV are shown. In C, fold increase in Kv inactivation percentage is expressed as a function of depolarizing voltage for WT control (black bars) or RIP-iPLA2β-TG islet β-cells (gray bars) at 10 min after switching medium glucose concentration from 0 to 20 mM.

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Ca2+/calmodulin-dependent protein kinase IIβ, and we have characterized the iPLA2β interactome further using

affinity capture and LC/electrospray ionization/MS/MS. An iPLA2β-FLAG fusion protein was expressed in an INS-1 insulinoma cell line and then adsorbed to an anti-FLAG matrix after cell lysis (13). iPLA2β and any associated proteins were then displaced with FLAG peptide and analyzed by SDS-PAGE. Gel sections were digested with trypsin, and the resultant peptide mixtures were analyzed by LC/MS/MS with database searching. This identified 37 proteins that associate with iPLA2β, and nearly half of them reside in ER or mitochondria. They include the ER chaperone calnexin, whose association with iPLA2β increases upon induction of ER stress. Phosphorylation of iPLA2β at Tyr616 also occurs upon induction of ER stress, and the phosphoprotein associates with calnexin. iPLA2β activity in vitro increases upon co-incubation with calnexin, and overexpress-sion of calnexin in INS-1 cells results in augmentation of ER stress-induced, iPLA2β-catalyzed hydrolysis of arachidonic acid from membrane phospholipids, reflecting the interaction’s functional significance. Similar results were obtained with mouse pancreatic islets (13). (J. Biol. Chem. 2010; 285: 33843-33857).

Fig. 47. Expression of FLAG-iPLA2β in INS-1 cells, followed by affinity capture, desorption, and SDS-PAGE analysis. INS-1 cells were stably transfected with lentivirus vector only (lanes 1 and 3) or with vector containing cDNA encoding FLAG-iPLA2β (lanes 2 and 4) with the tag at the C terminus. After incubation, cells were lysed, and the lysates were incubated with FLAG affinity resin and washed. Adsorbed proteins were then eluted and analyzed by 10% SDS-PAGE. Protein bands in lanes 1 and 2 were visualized with SYPRO Ruby stain. Lanes 3 and 4 represent immunoblots probed with antibody directed against iPLA2β.

Fig. 48. Tandem mass spectra of tryptic peptides from Ca2+-calmodulin-dependent protein kinase IIβ (A) and from calnexin (B) in LC-MS/MS analyses of digests of FLAG-iPLA2β and interacting proteins from INS-1 cells. FLAG-iPLA2β and interacting proteins were generated, isolated, digested, and analyzed by LC/MS/MS. (A) Tandem spectrum of 1 of 4 observed tryptic peptides from the Ca2+-calmodulin-dependent protein kinase IIβ sequence. (B) Tandem spectrum of 1 of 3 observed tryptic peptides from calnexin sequence.

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Fig. 50. Induction of ER stress with thapsigargin increases the association of calnexin with iPLA2β in INS-1 cells that overexpress FLAG-iPLA2β . INS-1 cells stably transfected to overexpress FLAG-iPLA2β were incubated for various intervals with 1 µM thapsigargin and then lysed. Lysates were processed to capture FLAG-iPLA2β and associated proteins, which were then analyzed by SDS-PAGE. (A) immunoblotting was performed with antibodies (Ab) against calnexin, and, after stripping and reprobing, for iPLA2β. (B) ris a plot of the densitometric ratios for calnexin over iPLA2β as a function of incubation time with thapsigargin.

Fig. 49. Immunochemical verification of identities of iPLA2β-interacting proteins identified by LC/MS/MS analyses. FLAG-iPLA2 and interacting proteins were generated in INS-1 cells, affinity-captured, and analyzed by SDS-PAGE. Immunoblotting was then performed with antibodies against calnexin (A) or SERCA (B).

Fig. 51. Effects of calnexin on Group VIA phospholipase A2 (iPLA2β) activity. The source of iPLA2β was the imidazole eluate from cobalt immobilized metal affinity columns onto which lysates of INS-1 cells that overexpress His-tagged iPLA2β had been applied. Calnexin (CNX) was prepared in a similar manner with INS-1 cells that overexpress His-calnexin. ATP (10 mM) and/or BEL (1 µM) were included in the incubation medium where indicated. Incubations were performed for 30 min at 37 °C, and PLA2 activity was measured as described. The p value for the difference between the conditions “iPLA2β” and “iPLA2β+CNX” is 0.0027, and that between “iPLA2β” and “iPLA2β+ATP” is 0.026.

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Fig. 52. Identification of a phosphotyrosine residue in iPLA2β after thapsigargin treatment of His-calnexin-INS1 cells. INS-1 cells were stably transfected to overexpress His-calnexin and incubated with thapsigargin or vehicle. The cells were then lysed, and the lysates were passed over cobalt affinity columns to capture and then elute His-calnexin and associated proteins, including iPLA2β. Eluates were processed by SDS-PAGE and tryptic digestion, and digests were analyzed by LC/MS/MS. A, tandem spectrum of a tryptic peptide (595FLDGGLLANNPTLDA-MTEIHEYNQDMIR62) from the iPLA2β sequence in which Tyr616 is phosphorylated that was obtained from materials in a thapsigargin-treated cell lysate. B, reconstructed ion chromatogram for the [M+3H]3+ ion (m/z 1091–1092) of that peptide from LC/MS analyses of tryptic digests. Solid line, thapsigargin-treated cells; dashed line, vehicle-treated cells. C, immunoblots from SDS-PAGE analyses of cobalt column eluates obtained from thapsigargin-treated cells. The eluates in lanes 1 and 3 (CONTROL) were not treated with phosphatase. The eluates in lanes 2 and 4 were treated with λ-protein phosphatase (λ-PPase) and protein phosphatase-1 (PP-1), respectively, before SDS-PAGE analyses. The blots were probed with antibody directed against iPLA2β.

Fig. 53. Effects of calnexin overexpression on ER stress-induced release of [3H]arachidonic acid from prelabeled cells. INS-1 cells stably transfected with a lentivirus vector construct that causes overexpression of His-calnexin (CNX-OE; dark bars) and cells transfected with empty vector (VECTOR, light bars) were prelabeled by incubation with [3H]arachidonic acid. To remove unincorporated radiolabel, the cells were incubated in serum-free medium and washed 3 times with glucose-free RPMI 1640 medium. Labeled cells were incubated in RPMI 1640 medium containing BEL (10 µM) or DMSO vehicle. After removal of medium, cells were placed in RPMI 1640 with 0.5% BSA that contained thapsigargin (THAPS; 1 µM) or vehicle (CONTROL or CON) and incubated. Cells were collected by centrifugation, and the supernatant 3H content was measured by liquid scintillation spectrometry. Amounts of released 3H were expressed as % incorporated 3H and normalized to the value for the vector control.

Fig. 54. Co-Immunoprecipitation of iPLA2β and calnexin from pancreatic islets isolated from mice. (A) Islets were isolated from C57BL/6J WT mice and incubated with thapsigargin. A lysate was then prepared in immunoprecipitation (IP) buffer containing 2% CHAPS and divided into 2 aliquots. One was incubated with Protein A-agarose and fetal bovine serum as a control (CON). The other was incubated with Protein A-agarose and a rabbit antibody (Ab) against iPLA2β to effect IP. The resultant immunoprecipitate was analyzed by SDS-PAGE, transferred to PVDF membrane, and probed with rabbit Ab against calnexin (upper panel). After stripping, the blot was probed with goat Ab against iPLA2β (middle panel). The lower panel represents a loading CON probed with anti-calnexin Ab. (B) Islets were isolated from RIP-iPLA2β-transgenic mice and lysed. The lysate was divided into 2 aliquots. One was incubated with Protein A-agarose and fetal bovine serum as CON. The other was incubated with Protein A-agarose and rabbit anti-calnexin Ab. The resultant immunoprecipitate was analyzed by SDS-PAGE, transferred to a PVDF membrane, and probed with T-14 iPLA2β antibody (upper panel). After stripping, the blot was probed with mouse anti-calnexin Ab (middle panel). The lower panel is a loading CON probed with T-14 iPLA2β Ab.

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B.5.) Group VIA phospholipase A2 (iPLA2β) is activated upstream of p38 MAP kinase in pancreatic islet beta cell signaling. 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 iPLA2 activity and amplified by iPLA2β overexpression. While exploring signaling events that occur downstream of iPLA2 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 (14). 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 -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

Fig. 55. Level of p38 MAPK phosphorylation increases in INS-1 cells and islets with iPLA2β expression level. Lysates from INS-1 cells or isolated pancreatic islets were analyzed by SDS-PAGE, transferred to PVDF membranes, and probed with antibody directed at phosphorylated p38 MAPK (A, upper bands) or total p38MAPK(A, lower bands). In B, the densitometric ratios of the bands for phospho-p38 and total p38 are plotted for stably transfected INS-1 cells that overexpress (OE) iPLA2β or for INS-1 cells transfected with vector only (Vector). In C, the densitometric ratios of the bands for phospho-p38 and total p38 are plotted for pancreatic islets isolated from transgenic (TG) mice that overexpress iPLA2β in β-cells or wild type (WT) mice.

Fig. 56. Secretagogue-induced insulin secretion from INS-1 insulinoma cells and pancreatic islets increases with iPLA2β expression level and is suppressed by pharmacologic inhibition of p38 MAPK. In A, pancreatic islets isolated from wild-type mice (WT; blue or yellow bars) or from transgenic mice (TG; red or green bars) that overexpress iPLA2β in islet β-cells were preincubated) with the p38 MAPK inhibitor PD169316 (20 µM) (yellow or green bars) or vehicle (blue or red bars) and then placed in fresh KRB mediumcontaining 0 or 20 mM D-glucose without or with 2.5 µM forskolin and incubated. In B, similar experiments were performed with INS-1 insulinoma cells stably transfected with empty vector (V; blue or yellow bars) or with a construct that causes overexpression of iPLA2β (OE; red or green bars) that were incubated without (blue or red bars) or with (yellow or green bars) PD169316 and with 0 or 20 mM D-glucose and 0 or 2.5 µM forskolin. At the end of the incubation period, aliquots of medium were removed for measurement of insulin.

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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 (14). (J. Biol. Chem. 2012; 287: 5528-5541.)

Fig. 57. Induction of ER stress with thapsigargin induces ceramide accumulation in INS-1 cells, and this is prevented by pharmacologic inhibition of p38 MAPK. INS-1 cells transfected with a construct that causes overexpression of iPLA2β (OE) or with empty vector (V) were preincubated with p38 MAPK inhibitor PD169316 (5 µM) or vehicle and then placed in fresh KRB medium without or with thapsigargin (1 µM). At the end of the incubation, cells were collected, homogenized, and extracted. The lipid extract was mixed with internal standard C8-ceramide, concentrated, reconstituted, infused in a solution with Li+ and analyzed by ESI/MS/MS scanning for constant neutral loss (CNL) of 48 from [M+Li]+ ions. Quantitation of ceramide (CM) species was achieved by dividing the ion current at the m/z value for that species by the ion current of the internal standard (m/z 432.5) and interpolating from a calibration curve. This value was normalized to lipid phosphorus. A and B, CNL spectra from INS-1 cells incubated with vehicle or 1 µM thapsigargin, respectively. In C & D, mean values for the normalized total amount of CM species are displayed for INS-1 cells transfected with empty vector (C) or that overexpress iPLA2β (D).

Fig. 58. Induction of ER stress with thapsigargin induces apoptosis of INS-1-OE cells, and this is prevented by pharmacologic inhibition of p38 MAPK. (A) Stably transfected INS-1 cells that overexpress iPLA2β were preincubated with the p38 MAPK inhibitor PD169316 (5 µM) or with diluent alone and then placed in fresh KRB medium without or with thapsigargin (1 µM). At the end of the incubation, cells were collected, stained with Annexin-V, and analyzed by FACS.

Fig. 59. The iPLA2β inhibitor BEL suppresses thapsigargin-induced p38 MAPK phosphoryla-tion in INS-1-OE cells, and this is reversed by exogenous arachidonic acid. INS-1 cells that overexpress iPLA2β were preincubated with BEL (10 µM) or vehicle alone. Then the medium was replaced by fresh medium containing 1 µM or no thapsigargin without or with BEL (10 µM) and without or with exogenous arachidonic acid (70 µM) and incubated. At the end of the incubation, cell lysates were prepared and analyzed by SDS-PAGE and immunoblotting with antibody against phos-phorylated p38 MAPK or total p38 MAPK (A). B displays mean values for densitometric ratios of bands for phospho-p38 and total p38. *, p < 0.05 for the value at the indicated condition vs. the condition in which incubation was performed in the presence of 1 µM thapsigargin, 10 µM BEL, and no exogenous arachidonic acid.

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Figure 60. Schematic diagram of signaling pathways in β-cells in which iPLA2β participates. Signals that activate iPLA2β in insulin-secreting β-cells include 1) incubation with D-glucose concentrations sufficient to stimulate insulin secretion and 2) incubation with SERCA inhibitors (e.g. thapsigargin) that deplete ER Ca2+ content, induce ER stress, and trigger β-cell apoptosis. iPLA2β activation occurs in signaling pathways involved both in D-glucose-stimulated insulin secretion and in SERCA inhibitor-induced apoptosis because both responses are attenuated by pharmacologic or genetic reductions in iPLA2β activity, and both are amplified by iPLA2β overexpression. Involvement of iPLA2β in insulin secre-tion may reflect effects of one of its products, arachidonic acid, to inhibit membrane Kv2.1 channel activity and to prolong the D-glucose-induced action potential in β-cells. Amplification of ceramide generation by iPLA2β may be a component of its parti-cipation in apoptosis. Data presented here indicate that p38 MAPK is activated during both glucose-stimulated insulin secretion and SERCA inhibitor-induced apoptosis in β-cells and that this occurs downstream of and requires prior iPLA2β activation. Intermediate events between iPLA2β activation and p38 MAPK activation include arachidonic acid release, its enzymatic oxygenation to bioactive eicosanoids (e.g. 12/15-lipoxygenase products), activation of Rho family small G-proteins (e.g. Rac1 and Cdc42), and activation of MAPK kinases (e.g. MEK3).

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B.6.) Group VIA Phospholipase A2 mitigates palmitate-induced β-cell mitochondrial injury and apoptosis. Palmitate (C16:0) induces apoptosis of insulin-secreting β-cells by processes that involve generation of reactive

oxygen species, and chronically elevated blood long chain free fatty acid levels may contribute to β-cell lipotoxicity and the development of diabetes mellitus. Group VIA phospholipase A2 (iPLA2β) affects β-cell sensitivity to apoptosis, and here we examined iPLA2β effects on events that occur in β-cells incubated with C16:0 (15). In INS-1 insulinoma cells these events included caspase-3 activation, stress response genes (C/EBP homologous protein and activating transcription factor 4) expression, ceramide accumulation, mitochondrial membrane potential loss, and apoptosis. All those responses were blunted in INS-1 cells that overexpress iPLA2β, which is proposed to facilitate repair of oxidized mitochondrial phospho-lipids, e.g. cardiolipin (CL), by excising oxidized polyunsaturated fatty acid resi-dues, e.g. linoleate (C18:2), to yield lyso-phospholipids, e.g. monolysocardiolipin (MLCL), that can be reacylated to regen-erate native phospholipid structures. Here mouse pancreatic islet MLCL was found to rise with increasing iPLA2β expression, and recombinant iPLA2β hydrolyzed CL to MLCL and released oxygenated C18:2 residues from oxidized CL in preference to native C18:2. C16:0 induced accumulation of oxidized CL species and the oxidized phospholipid (C18:0/hydroxyeicosatetraenoic acid)-glycerophosphoethanolamine, and these effects were blunted in INS-1 cells that overexpress iPLA2β, consistent with iPLA2β-mediated removal of oxidized phospholipids. C16:0 also induced iPLA2β association with INS-1 cell mitochondria, consistent with a role in mitochondrial repair (15). Thus, iPLA2β confers protection of β-cells against C16:0-induced injury. (J. Biol. Chem. 2014; 289:14194-210.)

Fig. 61. Incubation of INS-1 cells with palmitic acid induces apoptosis and this is attenuated by iPLA2β overexpression.

Fig. 62. C16:0-induced INS-1 cell iPLA2β mitochondrial association and loss of mitochondrial membrane potential (Δψm) is attenuated by iPLA2β overexpression. (A) Immunoblotting with iPLA2β and mitochondrial protein COX IV antibodies and (B) iPLA2β activity of INS-1 cell mitochondria. (Basal mitochondrial iPLA2 activity is attributable to the distinct iPLA2γ [235]). FACS of INS-1 cells loaded with JC1 indicator sensitive to Δψm and incubated with BSA (C) or C16:0 (D). E. C16:0-induced loss of Δψm in INS-1 cells transfected with vector only or that overexpress iPLA2β.

Fig. 63. Recombinant iPLA2β hydrolyzes cardiolipin to monolysocardiolipin (MLCL), and mouse islet MLCL content rises with iPLA2β expression level.

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Fig. 64. iPLA2β catalyzes hydrolysis of oxidized (C18:2)4-cardiolipin species to release oxygenated free fatty acids (FFA). A is the negative ion ESI/MS spectrum of standard (C18:2)4-CL and shows the [M-H]- ion and 13C isotopomers and little signal from m/z 1460 to 1600. B is a spectrum after oxidation with cytochrome c/H2O2 that shows formation of oxidation products reflected by peaks with m/z 1448+(nx16) where n = 1–8. C is the ESI/MS spectrum from m/z 250 to 320 of the lipid extract of an incubation mixture of partially oxidized CL and His-iPLA2β and shows [M-H]- ions of internal standard [2H4]palmitate (d4-C16:0,m/z 259), linoleate (C18:2,m/z 279), oxylinoleate (O-C18:2, m/z 295), and dioxylinoleate (O2-C18:2, m/z 311). D is the time course of iPLA2β-catalyzed FFA release from oxidized CL.

Fig. 65. Palmitate induces accumulation of an oxy-cardiolipin species in β -cells, and this is attenuated by overexpression of iPLA2β.

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C.) Group VIB Phospholipase A2 (iPLA2γ): Mice deficient in Group VIB Phospholipase A2 exhibit relative resistance to obesity and metabolic abnormalities induced by a Western Diet. Phospholipases A2 (PLA2) play important roles in metabolic processes, and the Group VI PLA2 family is comprised of intracellular enzymes

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that do not require Ca2+ for catalysis. Mice deficient in Group VIA PLA2 (iPLA2β) develop more severe glucose intolerance than wild-type (WT) mice in response to dietary stress. Group VIB PLA2 (iPLA2γ) is a related enzyme distributed in membranous organelles, including mitochondria, and iPLA2γ knockout (KO) mice exhibit altered mitochondrial morphology and function. We have compared metabolic responses of iPLA2γ-KO and

Fig. 70. ESI-MS of acylcarnitine molecular species in gastrocnemius muscle from iPLA2γ-KO & WT mice fed SC or WD. Acylcarnitines in homogenates of gastrocnemius muscle from 6 mo WT (A & B) and iPLA2γ-KO (C & D) male mice fed SC (A & C) or WD (B & D) were analyzed as methyl esters relative to [2H3]acetylcarnitine internal standard by positive ion ESI/MS/MS scans.

Fig. 71. Acylcarnitine content and mean chain length in gastrocnemius muscle & liver of iPLA2γ-KO and WT mice fed SC or WD. Acylcarnitines in homogenates of gastrocnemius muscle (A & B) and liver (C & D) from 6 mo WT (light bars) and iPLA2γ-KO (dark bars) male mice fed SC or WD were quantitated by ESI/MS/MS. A and C: sum of moles of acyl chain carbon for endogenous long-chain acylcarnitine (LCAC) species/mg tissue protein normalized to value for WT mice fed SC. B and D: mean carbon chain length computed from the ratio moles of acyl chain carbon relative to moles of acylcarnitine. (*) (P<0.05) in genotype comparisons; (x), (P< .05) in diet comparisons.

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WT mice fed a Western diet (WD) with a high fat content (16). 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 β-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 (16). The iPLA2γ-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. D.) Literature Cited Mass Spectrometry of Lipid Antigens and Products of Lipid Metabolizing

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