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BBA - Molecular and Cell Biology of Lipids (v.1781, #5)

Editorial Board (pp. ii).

Lipid peroxidation and decomposition — Conflicting roles in plaque vulnerability and stability by Sampath Parthasarathy; Dmitry Litvinov; Krithika Selvarajan; Mahdi Garelnabi (pp. 221-231).

The low density lipoprotein (LDL) oxidation hypothesis has generated considerable interest in oxidative stress and how it might affect atherosclerosis. However, the failure of antioxidants, particularly vitamin E, to affect the progression of the disease in humans has convinced even staunch supporters of the hypothesis to take a step backwards and reconsider alternatives. Preponderant evidence for the hypothesis came from animal antioxidant intervention studies. In this review we point out basic differences between animal and human atherosclerosis development and suggest that human disease starts where animal studies end. While initial oxidative steps in the generation of early fatty streak lesions might be common, the differences might be in the steps involved in the decomposition of peroxidized lipids into aldehydes and their further oxidation into carboxylic acids. We suggest that these steps may not be amenable to attenuation by antioxidants and antioxidants might actually counter the stabilization of plaque by preventing the formation of carboxylic acids which are anti-inflammatory in nature. The formation of such dicarboxylic acids may also be conducive to plaque stabilization by trapping calcium. We suggest that agents that would prevent the decomposition of lipid peroxides and promote the formation and removal of lipid hydroxides, such as paraoxonase (PON 1) or apo A1/high density lipoprotein (HDL) might be more conducive to plaque regression.

Keywords: Atherosclerosis; Aldehyde; Dicarboxylation; Inflammation; Calcification

Recycling of apolipoprotein E is not associated with cholesterol efflux in neuronal cells by Lars Rellin; Joerg Heeren; Ulrike Beisiegel (pp. 232-238).

After receptor-mediated endocytosis of apolipoprotein E (apoE)-containing lipoproteins in hepatocytes, the isoform apoE3 is efficiently recycled in a process which is associated with cholesterol efflux. Recycling and cholesterol efflux are greatly reduced when apoE4 is the only isoform present. ApoE is the main apolipoprotein in cerebrospinal fluid, and it plays a pivotal role in maintaining cholesterol homeostasis in the brain. The isoform apoE4 is associated with an increased risk of Alzheimer's disease and it has been postulated that high intracellular cholesterol levels promote the amyloidogenic processing of amyloid precursor protein. Therefore we investigated the cellular processing of different apoE isoforms as well as the associated cholesterol efflux in the murine neuronal cell line HT-22. Uptake of apoE3-containing lipoproteins resulted in the expected recycling while, as seen in non-neuronal cells, recycling of apoE4 was significantly reduced. However, despite these differences in apoE recycling, there was no difference in rates of cholesterol efflux. Therefore we conclude that in this neuronal cell model the reduced recycling of apoE4 does not affect cellular cholesterol metabolism.

Keywords: Abbreviations; AD; Alzheimer's disease; Apo; apolipoprotein; Aβ; Amyloidβ; APP; amyloid precursor protein; ABCA1; ABCG1, ATP binding cassette transporter A1/G1; BSA; bovine serum albumin; CSF-Lp; cerebrospinal fluid lipoproteins; FCS; fetal calf serum; HDL; high density lipoproteins; HT-22 cells; murine neuroblastoma cell line; LDL; low density lipoproteins; LDLR; low density lipoprotein receptor; LRP1; low density lipoprotein receptor-related protein 1; PBS; phosphate-buffered saline; PFA; paraformaldehyde; SR-BI; scavenger receptor BI; TRL; triglyceride-rich lipoproteins; VLDL; very low density lipoproteinsApolipoprotein E; Cholesterol; Lipoprotein; Endocytosis; Alzheimer's disease

Involvement of ZIP/p62 in the regulation of PPARα transcriptional activity by p38-MAPK by Claire Diradourian; Cédric Le May; Cauzac Michèle Caüzac; Jean Girard; Anne-Françoise Burnol; Pegorier Jean-Paul Pégorier (pp. 239-244).

The peroxisome proliferator-activated receptor alpha (PPARα) belongs to the nuclear receptor family and plays a central role in the regulation of lipid metabolism, glucose homeostasis and inflammatory processes. In addition to its ligand-induced activation, PPARα is regulated by phosphorylation via ERK-MAPK, PKA and PKC. In this study we examined the effect of p38-MAPK on PPARα transcriptional activity. In COS-7 cells, anisomycin, a p38 activator, induced a dose-dependent phosphorylation of PPARα and a 50% inhibition of its transcriptional activity. In H4IIE hepatoma cells, anisomycin-induced p38 phosphorylation decreased both endogenous and PPARα ligand-enhanced L-CPTI and ACO gene expression. Interestingly, PPARα/p38 interaction required the molecular adapter ZIP/p62. Reducing ZIP/p62 expression by siRNA, partially reversed the inhibitory effect of anisomycin on L-CPTI gene expression. In conclusion, we showed that p38 activation induced PPARα phosphorylation and inhibition of its transcriptional activity through a trimeric interaction between p38-MAPK, ZIP/p62 and PPARα.

Keywords: PPARα; p38-MAPK; ZIP/p62; L-CPTI; Phosphorylation; Anisomycin; Reporter gene; Co-immunoprecipitation; siRNA; Hepatoma cell

Cholesterol is a determinant of the structures of discoidal high density lipoproteins formed by the solubilization of phospholipid membranes by apolipoprotein A-I by John B. Massey; Henry J. Pownall (pp. 245-253).

Formation of discoidal high density lipoproteins (rHDL) by apolipoprotein A-I (apoA-I) mediated solubilization of dimyristoyl phosphatidylcholine (DMPC) multilamellar vesicles (MLV) was dramatically affected by bilayer cholesterol concentration. At a low ratio of DMPC/apoA-I (2 mg DMPC/mg apoA-I, 84/1 mol/mol), sterols (cholesterol, lathosterol, and β-sitosterol) that form ordered lipid phases increase the rate of solubilization similarly, yielding rHDL with similar structures. By changing the temperature and sterol concentration, the rates of solubilization varied almost 3 orders of magnitude; however, the sizes of the rHDL were independent of the rate of their formation and dependent upon the bilayer sterol concentration. At a high ratio of DMPC/apoA-I (10/1 mg DMPC/mg apoA-I, 420/1 mol/mol), changing the temperature and cholesterol concentration yielded rHDL that varied greatly in size, phospholipid/protein ratio, mol% cholesterol, and number of apoA-I molecules per particle. rHDL were isolated that had 2, 4, 6, and 8 molecules of apoA-I per particle, mean diameters of 117, 200, 303, and 396 Å, and a mol% cholesterol that was similar to the original MLV. Kinetic studies demonstrated that the different sized rHDL are formed independently and concurrently. The rate of formation, lipid composition, and three-dimensional structures of cholesterol-rich rHDL is dictated primarily by the original membrane phase properties and cholesterol content. The size speciation of rHDL and probably nascent HDL formed via the activity of the ABCA1 lipid transporter is mechanistically linked to the cholesterol content of the membranes from which they were formed.

Keywords: Abbreviations; ABCA1; ATP binding cassette transporter A1; ApoA-I; apolipoprotein A-I; DMPC; dimyristoyl-; sn; -glycero-3-phosphocholine; DPH; 1,6-diphenyl-1,3,5-hexatriene; DPPC; dipalmitoyl-; sn; -glycero-3-phosphocholine; DSC; differential scanning calorimetry; EM; transmission electron microscopy; HDL; high density lipoproteins; l; _d; liquid-disordered phase; l; _o; liquid-ordered phase; LUV; large unilamellar vesicles; MLV; multilamellar vesicles; mol/mol; molar ratio of DMPC/apoA-I; PAGE; polyacrylamide gel electrophoresis; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; rHDL; reassembled discoidal HDL; RCT; reverse cholesterol transport; s; _o; solid-ordered or gel phase; SM; sphingomyelin; T; _m; midpoint temperature of the gel to; l; _d; phase transitionReverse cholesterol transport; apoA-I; Cholesterol; High density lipoprotein; Phosphatidylcholine; Microsolubilization

Characterization of the murine CTP:phosphocholine cytidylyltransferase beta gene promoter by Hebe Marcucci; Claudia Elena; Paola Gilardoni; Claudia Banchio (pp. 254-262).

CTP:phosphocholine cytidylyltransferase (CCT) is a key regulatory enzyme in phosphatidylcholine (PtdCho) biosynthesis by the Kennedy pathway. In mammals, there are two genes that encode the enzyme isoforms that catalyze this reaction: Pcyt1a for CCTα and Pcyt1b for CCTβ. In mouse tissues two different CCTβ variants named CCTβ2 and CCTβ3 have been identified. Although little is known about Pcyt1b gene expression, recent data from cell lines propose a distinct role for CCTβ2 in neuronal differentiation. Also, gonadal dysfunction in the CCTβ2 knockout mouse suggests a role for this protein in ovary maturation and the maintenance of sperm production. This work defines and characterizes two alternative promoters that drive the expression of the two murine CCTβ isoforms. The promoter activities were measured in Neuro-2a (mouse neuroblastoma), TM4 (mouse Sertoli) and C3H10T1/2 (mouse embryo fibroblast) cell lines. The transcriptional start points of each transcript and the promoter regions essential for the expression of each isoform were determined. Analysis of the CCTβ2 promoter sequence suggested the transcription factor AP-1 as a potential regulator of CCTβ2 expression in neuronal cells. However, CCTβ3 was not detected in this cell line suggesting a different role or regulation. The activities of alternative promoters provide for greater flexibility in the control of CCTβ isoform expression.

Keywords: Abbreviations; PtdCho; phosphatidylcholine; CCT; CTP:phosphocholine cytidylyltransferas; FBS; fetal bovine serum; Luc; luciferase; TF; transcriptional factor Pcyt1b; gene promoter; Cytidylyltransferase; Phosphatidylcholine; Brain; Testis

Potential role of phospholipase D2 in increasing interleukin-2 production by T-lymphocytes through activation of mitogen-activated protein kinases ERK1/ERK2 by Safouane M. Hamdi; Clotilde Cariven; Sophie Coronas; Nicole Malet; Hugues Chap; Bertrand Perret; Jean-Pierre Salles; Michel Record (pp. 263-269).

Hydrolysis of phosphatidylcholine by phospholipase D (PLD) leads to the generation of phosphatidic acid (PA), which is itself a source of diacylglycerol (DAG). These two versatile lipid second messengers are at the centre of a phospholipid signalling network and as such are involved in several cellular functions. However, their role in T-cell activation and functions are still enigmatic. In order to elucidate this role, we generated a human and a murine T-cell line that stably overexpressed the PLD2 isoform. Analysis of the Ras–MAPK pathway upon phorbol myristate acetate (PMA) and ionomycin stimulation revealed that PLD2 promoted an early and sustained increase in ERK1/2 phosphorylation in both cell lines. This response was inhibited by 1-butanol, a well known distracter of PLD activity, or upon overexpression of a dominant negative PLD2, and it was concomitant with a boost of PA/DAG production. As a functional consequence of this PLD2-dependent MAPK activation, interleukin-2 production evoked by PMA/ionomycin stimulation or CD3/CD28 engagement was enhanced in the two T-cell lines overexpressing PLD2. Thus, PLD2 emerged as an early player upstream of the Ras-MAPK-IL-2 pathway in T-cells via PA and DAG production, raising new possibilities of pharmacological manipulation in immune disorders.

Keywords: Abbreviations; PLD2; phospholipase D2; PA; phosphatidic acid; DAG; diacylglycerol; PC; phosphatidylcholine; TCR; T-cell receptor; IL-2; interleukin-2; MAPK; mitogen-activated protein kinase; ERK; extracellular signal-regulated kinase; LFA-1; lymphocyte function-associated antigen-1; PAP; phosphatidate phosphohydrolasePhospholipase D2; Phosphatidic acid; Diacylglycerol; T-lymphocytes; Interleukin-2; Mitogen-activated protein kinases (MAPK)

Increased production of the ether-lipid platelet-activating factor in intestinal epithelial cells infected by Salmonella enteritidis by Laia Egea; Gimenez Rosa Giménez; Lucia David Lúcia; Ines Modolell; Badia Josefa Badía; Laura Baldoma; Juan Aguilar (pp. 270-276).

When exposed to enteric pathogens intestinal epithelial cells produce several cytokines and other proinflammatory mediators. To date there is no evidence that the ether-lipid platelet-activating factor (PAF) is one of these mediators. Our results revealed a significant increase in PAF production by human colonic tissue 4 h after infection by enterohemorrhagic Escherichia coli (EHEC) or Salmonella enteritidis. PAF is produced in the gut by cells of the immune system in response to bacterial infection. To determine whether the epithelial cells of colonic mucosa might also modulate PAF levels, we carried out PAF quantification and analysis of the enzymes involved in PAF synthesis in 5-day-old (undifferentiated) or 28-day-old (differentiated) Caco-2 cell cultures. Infection of undifferentiated Caco-2 cells with either bacterium had no effect on PAF levels, whereas in differentiated cells, infection by S. enteritidis increased PAF levels.Following infection by S. enteritidis, there were no changes in the activity of dithiothreitol-insensitive choline phosphotransferase. However, the enzymes of the remodeling pathway cytosolic phospholipase A₂, which catalyzes the formation of the PAF precursor lysoPAF, and lysoPAF acetyltransferase, are activated in the infected epithelial cells. This response is Ca²⁺-dependent.

Keywords: Abbreviations; BAPTA/AM; N; ,; N; ,; N; ′,; N; ′-tetraacetic acid tetra(acetoxymethyl)ester; CPT; dithiothreitol-insensitive choline phosphotransferase; DIC; differential interface contrast; DMEM; Dulbecco's Modified Eagle Medium; DTT; dithiothreitol; EGTA; ethylene glycol tetraacetic acid; EHEC; enterohemorrhagic; Escherichia coli; FAS; fluorescence actin staining; FITC; fluorescein isothiocyanate; LB; Luria–Bertani broth; LDH; lactate dehydrogenase; lysoPAF-AT; acetyl-coenzyme A: lysoPAF acetyltransferase; MAFP; methyl arachidonylfluorophosphonate; PAF; platelet-activating factor; PAF-AH; PAF-acetylhydrolase; cPLA; ₂; cytosolic phospholipase A; ₂; iPLA; ₂; Ca; ²⁺; -independent PLA; ₂; Pyrrolidine-1; N; -{(2S,4R)-4-(biphenyl-2-ylmethyl-isobutyl-amino)-1-[2-(2,4-difluorobenzoyl)-benzoyl]-pyrrolidin-2-ylmethyl}-3-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)-phenyl]acrylamide, HClEther-lipid metabolism; Platelet-activating factor; Cytosolic phospholipase A; ₂; Gut inflammation; Enterohemorrhagic; Escherichia coli; Salmonella enteritidis

Protein kinase CβI interacts with the β1-adrenergic signaling pathway to attenuate lipolysis in rat adipocytes by Jiro Nakamura (pp. 277-281).

We have shown previously that insulin attenuates β1-adrenergic receptor (β1-AR)-mediated lipolysis via activation of protein kinase C (PKC) in rat adipocytes. This antilipolysis persists after removal of insulin and is independent of the phosphodiesterase 3B activity, and phorbol 12-myristate 13-acetate (PMA) could substitute for insulin to produce the same effect. Here, we attempted to identify the PKC isoform responsible for antilipolysis. Isolated adipocytes were treated with high and low concentrations of PMA for up to 6 h to degrade specific PKC isoforms. In the PMA-treated cells, the downregulation profiles of PKC isoforms α and βI, but not βII, δ, ɛ, or ζ, correlated well with a decrease of lipolysis-attenuating effect of PMA. After rats fasted for 24 h, adipocyte expression of PKC isoform α increased, while expression of PKCδ decreased. Fasting did not change the potency of PMA to attenuate lipolysis, however. The lipolysis-attenuating effect of PMA was blocked by the PKCβI/βII inhibitor LY 333531, but not by the PKCβII inhibitor CGP 53353 or the PKCδ inhibitor rottlerin. These data suggest that PKCβI interacts with β1-AR signaling and attenuates lipolysis in rat adipocytes.

Keywords: Abbreviations; β-AR; β-adrenergic receptor; PKA; protein kinase A; PKB; protein kinase B; PKC; protein kinase C; cPKC; conventional PKC; nPKC; novel PKC; aPKC; atypical PKC; PI3K; phosphatidylinositol 3-kinase; HSL; hormone-sensitive lipase; PDE3B; phosphodiesterase 3B; IRS; insulin-receptor substrate; PMA; phorbol 12-myristate 13-acetate; DAG; diacylglycerol; HEPES; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; BSA; bovine serum albumin; SDS; sodium dodecylsulphate; buffer A; Dulbecco's modified Eagle's medium supplemented with 20 mM HEPES, pH 7.4, 20 mg/ml BSA, and 200 nM adenosine; buffer B; 119 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 2.6 mM CaCl2, 1.2 mM MgSO4, and 32.3 mM HEPES, pH 7.4, 2 mM glucose, 20 mg/ml BSA, and 200 nM adenosine; buffer C; 10 mM Tris, pH 7.4, 0.15 M NaCl, 1 mM EDTA, and 1 mM EGTAAdipocyte; Lipolysis; β-Adrenergic receptor; PKC; PKCβI

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BBA - Molecular and Cell Biology of Lipids (v.1781, #5)