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BBA - Molecular and Cell Biology of Lipids (v.1771, #4)

Editorial Board (pp. ii).

Lipid composition of peroxisomes from the yeast Pichia pastoris grown on different carbon sources by Tamara Wriessnegger; Georg Gübitz; Erich Leitner; Elisabeth Ingolic; James Cregg; Bernard J. de la Cruz; Günther Daum (pp. 455-461).

Highly purified peroxisomes from the yeast Pichia pastoris grown on methanol or oleic acid, respectively, were used to characterize the lipid composition of this organelle. For this purpose, an isolation procedure had to be adapted which yielded highly purified P. pastoris peroxisomes. When peroxisome proliferation was induced by growth on methanol, alcohol oxidase was the predominant peroxisomal protein. Cultivation of P. pastoris on oleic acid led to induction of a family of peroxisomal enzymes catalyzing fatty acid β-oxidation, whose most prominent members were identified by mass spectrometry. On either carbon source, phosphatidylcholine and phosphatidylethanolamine were the major peroxisomal phospholipids, and cardiolipin was present in peroxisomal membranes at a substantial amount, indicating that this phospholipid is a true peroxisomal component. Ergosterol was the most abundant sterol of P. pastoris peroxisomal membranes irrespective of the culture conditions. The fatty acid composition of whole cells and peroxisomes was highly affected by cultivation of P. pastoris on oleic acid. Under these conditions, oleic acid became the predominant fatty acid in phospholipids from total cell and peroxisomal extracts. Thus, oleic acid was not only utilized as an appropriate carbon source but also as a building block for complex membrane lipids. In summary, our data provide first insight into biochemical properties of P. pastoris peroxisomal membranes, which may become important for the biotechnological use of this yeast.

Keywords: Abbreviations; AOX; alcohol oxidase; TLC; thin layer chromatography; GLC; gas–liquid chromatography; LP; lysophospholipid; PtdSer; phosphatidylserine; PtdIns; phosphatidylinositol; PtdOH; phosphatidic acid; PtdCho; phosphatidyl choline; PtdEtn; phosphatidylethanolamine; CL; cardiolipin; DM-PtdEtn; dimethylphosphatidyl ethanolamine Pichia pastoris; Peroxisomes; Alcohol oxidase; Phospholipids; Sterol; Oleic acid

Diacylglycerol kinase α suppresses tumor necrosis factor-α-induced apoptosis of human melanoma cells through NF-κB activation by Kenji Yanagisawa; Satoshi Yasuda; Masahiro Kai; Shin-ichi Imai; Keiko Yamada; Toshiharu Yamashita; Kowichi Jimbow; Hideo Kanoh; Fumio Sakane (pp. 462-474).

We investigated the implication of diacylglycerol kinase (DGK) α (type I isoform) in melanoma cells because we found that this DGK isoform was expressed in several human melanoma cell lines but not in noncancerous melanocytes. Intriguingly, the overexpression of wild-type (WT) DGKα, but not of its kinase-dead (KD) mutant, markedly suppressed tumor necrosis factor (TNF)-α-induced apoptosis of AKI human melanoma cells. In the reverse experiment, siRNA-mediated knockdown of DGKα significantly enhanced the apoptosis. The overexpression of other type I isoforms (DGKβ and DGKγ) had, on the other hand, no detectable effects on the apoptosis. These results indicate that DGKα specifically suppresses the TNF-α-induced apoptosis through its catalytic action. We found that the overexpression of DGKα-WT, but not of DGKα-KD, further enhanced the TNF-α-stimulated transcriptional activity of an anti-apoptotic factor, NF-κB. Conversely, DGKα-knockdown considerably inhibited the NF-κB activity. Moreover, an NF-κB inhibitor blunted the anti-apoptotic effect of DGKα overexpression. Together, these results strongly suggest that DGKα is a novel positive regulator of NF-κB, which suppresses TNF-α-induced melanoma cell apoptosis.

Keywords: Diacylglycerol kinase; Apoptosis; Tumor necrosis factor-α; NF-κB; Melanoma

Differences in cell morphology, lipid and apo B secretory capacity in caco-2 cells following long term treatment with saturated and monounsaturated fatty acids by Paul A. Bateman; Kim G. Jackson; Vatsala Maitin; Parveen Yaqoob; Christine M. Williams (pp. 475-485).

The suitability of the caco-2 cell line as a model for studying the long term impact of dietary fatty acids on intestinal lipid handling and chylomicron production was examined. Chronic supplementation of caco-2 cells with palmitic acid (PA) resulted in a lower triacylglycerol secretion than oleic acid (OA). This was coupled with a detrimental effect of PA, but not OA, on transepithelial electrical resistance (TER) measurements, suggesting a loss of structural integrity across the cell monolayer. Addition of OA reversed the adverse effects of PA and stearic acid on TER and increased the ability of cells to synthesise and accumulate lipid, but did not normalise the secretion of lipids by caco-2 cells. Increasing amounts of OA and decreasing amounts of PA in the incubation media markedly improved the ability of cells to synthesise apolipoprotein B and secrete lipids. Real time RT-PCR revealed a down regulation of genes involved in lipoprotein synthesis following PA than OA. Electron microscopy showed adverse effects of PA on cellular morphology consistent with immature enterocytes such as stunted microvilli and poor tight junction formation. In conclusion, previously reported differences in lipoprotein secretion by caco-2 cells supplemented with saturated fatty acids (SFA) and OA may partly reflect early cytotoxic effects of SFA on cellular integrity and function.

Keywords: Apolipoprotein B; Monounsaturated fatty acid; Saturated fatty acid; Real time RT-PCR; Taurocholate micelle; Tight junction

Choline cannot be replaced by propanolamine in mice by Zhaoyu Li; Dennis E. Vance (pp. 486-490).

Choline is an important nutrient for humans and animals. Animals obtain choline from the diet and from the catabolism of phosphatidylcholine made by phosphatidylethanolamine N-methyltransferase (PEMT). The unique model of complete choline deprivation is Pemt^−/− mice that are fed a choline-deficient diet. This model, therefore, can be used for the examination of choline substitutes in mammalian systems. Recently, propanolamine was found to be a replacement for choline in yeast. Thus, we tested to see whether or not choline can be replaced by propanolamine in mice. Mice were fed a choline-deficient diet and supplemented with either methionine, 2-amino-propanol, 2-amino-isopropanol and 3-amino-propanol. We were unable to detect the formation of any of the possible phosphatidylpropanolamines. Moreover, none of them prevented liver damage, reduction of hepatic phosphatidylcholine levels or fatty liver induced in choline-deficient- Pemt− /⁻ mice. These results suggest that choline in mice cannot be replaced by any of the three propanolamine derivatives.

Keywords: Abbreviations; CD; choline-deficient; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PEMT; phosphatidylethanolamine; N; -methyltransferase; ALT; alanine aminotransferases; TG; triacylglycerol; AdoMet; S; -adenosylmethinonineCholine; Phosphatidylethanolamine; N; -methyltransferase; Phosphatidylcholine; Propanolamine; Choline-deficient

Production of bioactive lysophosphatidic acid by lysophospholipase D in hen egg white by Junichi Morishige; Kanako Touchika; Tamotsu Tanaka; Kiyoshi Satouchi; Kenji Fukuzawa; Akira Tokumura (pp. 491-499).

Lysophosphatidic acid (LPA), a lysophospholipid mediator, is produced extracellularly by lysophospholipase D (lysoPLD) secreted in several animal body fluids including blood plasma. Previously, we reported that hen egg white contains polyunsaturated fatty acid-rich LPA. In this study, we examined whether lysoPLD is involved in the production of LPA in hen egg white. LysoPLD activity was measured by determining LPA and choline by mass spectrometric and enzyme-linked fluorometric analyses, respectively. LysoPLD increased with increased dilution of egg white, indicating that one or more components of egg white strongly inhibit its lysoPLD activity. This dilution-dependent increase in the lysoPLD activity was masked by co-incubation of the egg white with lysozyme, a major protein in hen egg white. Furthermore, addition of Zn²⁺, Mn²⁺, Ni²⁺, or Co²⁺ to diluted egg white altered preference patterns of lysoPLD toward choline-containing substrates. In particular, the egg white lysoPLD activity was greatly increased when Co²⁺ was added. The cation-requirement of lysoPLD activity in hen egg white resembled that of plasma autotaxin (ATX)/lysoPLD. Western blot analysis revealed that egg white contained a protein that was immunostained with anti-ATX antibody. These results suggested that LPA in hen egg white is produced from lysophospholipids, especially LPC, by the action of ATX/lysoPLD, possibly originating from hen oviduct fluid.

Keywords: Abbreviations; ATX; autotaxin; BSA; bovine serum albumin; GPC; l; -α-glycerophosphorylcholine; LPA; lysophosphatidic acid; LPC; lysophosphatidylcholine; lysoPLD; lysophospholipase D; NPP; ecto-nucleotide pyrophosphatase/phosphodiesterase; PC; phosphatidylcholine; SPC; sphingosylphosphorylcholine; TLC; thin-layer chromatography; MALDI-TOF-MS; matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Fatty acyl moieties of phospholipids are designated in terms of the number of carbon atoms: the number of double bondsAutotaxin; Choline; Egg white; Lysophosphatidic acid; Lysophospholipase D; Lysozyme

Activation of caspase 3 in HepG2 cells by elaidic acid (t18:1) by Yumi Kondoh; Teruo Kawada; Reiko Urade (pp. 500-505).

In order to examine the effects of trans-unsaturated fatty acids (TFAs) on HepG2 cells, cells were grown in serum-free media supplemented with elaidic acid (t18:1); t18:1 is the trans-isomer of oleic acid and is the major component of TFAs in foods. Both t18:1 and palmitic acids (16:0) at concentrations higher than 100 μM inhibited growth and decreased the rate of protein synthesis. The presence of phosphatidylserine in the outer leaflet of the lipid bilayer, indicative of apoptosis, occurred 1 h after the addition of both t18:1 and 16:0 to the media. Caspase 3 was found to be activated by these fatty acids: caspase 8 was activated by 16:0 and only moderately by t18:1. Activation of caspase 3 by these fatty acids was fully inhibited by a caspase 8 inhibitor. However, growth inhibition by t18:1 was partially prevented by the caspase 8 inhibitor. These results suggest that cell death caused by t18:1 may proceed by both caspase-dependent and -independent pathways.

Keywords: Elaidic acid; Palmitic acid; HepG2; Apoptosis; Caspase

Defect in fatty acid esterification of dolichol in Niemann–Pick type C1 mouse livers in vivo by Mikael Turunen; Sophia Schedin-Weiss (pp. 506-513).

Fatty acid esterification of dolichol and cholesterol in Niemann–Pick type C1 mouse (Balb/c NIH npc1^−/−) livers was investigated in response to treatment with peroxisomal proliferators. These inducers have hypolipidemic properties and influence the mevalonate pathway and the intracellular transport of the final products of this biosynthetic route. Such inducers are consequently interesting to use in a disease model with defective intracellular transport of lipids. In wild-type mice, the levels of dolichol and cholesterol found as free alcohols were not changed to any great extent upon treatment with the peroxisomal inducers dehydroepiandrosterone, clofibrate and diethylhexylphtalate. In contrast, the amounts of dolichyl esters increased whereas cholesteryl esters decreased by the same treatments. The rate of enzymatic esterification of dolichol in isolated microsomes was accordingly elevated after 5- to 7-day treatments with the efficient peroxisomal proliferators DEHP and PFOA, while the corresponding esterification of cholesterol was decreased. Upon peroxisomal induction in npc1^−/− mice, the enzymatic dolichol esterification in vitro increased whereas the low concentration of dolichyl esters remained unchanged. The results thus demonstrate that the induction of fatty acid esterification of dolichol in vivo is impaired in npc1^−/− mouse liver. It is therefore proposed that the intracellular lipid transport defect in npc1^−/− mouse liver disables either dolichol and/or the fatty acid from reaching the site of esterification in vivo. This proposal was strengthened by the finding that the amount of dolichol was decreased in an isolated Golgi fraction from npc1^−/− mice.

Keywords: Abbreviations; NPC; Niemann–Pick type C disease; ADAT; acyl-coenzyme A dolichol acyltransferase; ACAT; acyl-coenzyme A cholesterol acyltransferase; dolichyl-P; dolichyl monophosphate; CPT; cis; -prenyltransferase; DEHP; diethylhexylphtalate; PFOA; perfluorooctanoic acid; C:M; chloroform:methanol; C:M:W; chloroform:methanol:waterDolichol; Dolichyl ester; Cholesteryl ester; Peroxisomal proliferation; Niemann–Pick type C disease; Lipid transport

Two waves of the nuclear phospholipase C activity in serum-stimulated HL-60 cells during G₁ phase of the cell cycle by Vesna Lukinovic-Skudar; Katarina Matkovic; Hrvoje Banfic; Dora Visnjic (pp. 514-521).

Phosphatidylinositol-specific phospholipase C (PI-PLC) is activated in cell nuclei during the cell cycle progression. We have previously demonstrated two peaks of an increase in the nuclear PI-PLC activities in nocodazole-synchronized HL-60 cells. In this study, the activity of nuclear PI-PLC was investigated in serum-stimulated HL-60 cells. In serum-starved HL-60 cells, two peaks of the activity of nuclear PI-PLC were detected at 30 min and 11 h after the re-addition of serum with no parallel increase in PLC activity in cytosol, postnuclear membranes or total cell lysates. An increase in the serine phosphorylation of b splicing variant of PI-PLCβ₁ was detected with no change in the amount of PI-PLCβ_1b in nuclei isolated at 30 min and 11 h after the addition of serum. PI-PLC inhibitor ET-18-OCH₃ and MEK inhibitor PD 98059 completely abolished serum-mediated increase at both time-points. The addition of inhibitors either immediately or 6 h after the addition of serum had inhibitory effects on the number of cells entering S phase. These results demonstrate that two waves of nuclear PI-PLCβ_1b activity occur in serum-stimulated cells during G₁ phase of the cell cycle and that the later increase in the PLC activity is equally important for the progression into the S phase.

Keywords: Phospholipase Cβ; _1b; Nuclei; Serum; Cell cycle; G; ₁; phase; HL-60 cells

Activation of cytosolic phospholipase A₂ and 15-lipoxygenase by oxidized low-density lipoproteins in cultured human lung fibroblasts by Gabriella Lupo; Carmelina Daniela Anfuso; Nicolò Ragusa; Cataldo Tirolo; Bianca Marchetti; Elisa Gili; Cristina La Rosa; Carlo Vancheri (pp. 522-532).

In cell cultures of human lung fibroblasts, we found that oxidized LDL (oxLDL), after 24-h treatment, stimulated arachidonic acid release. A putative role for phospholipases A₂ and MAPK activities in this process was postulated. Consequently, we studied the contribution of either Ca²⁺-dependent, cytosolic phospholipase A₂ (cPLA₂) or Ca²⁺-independent phospholipase A₂ (iPLA₂), and the role of the MAP kinase family in oxLDL toxicity to fibroblastic cells in vitro. Activation of extracellular signal-regulated kinases ERK1/2, p38 and c-Jun NH₂-terminal kinase (JNK) was also assessed with Western blotting. Compared with cellular samples untreated or treated with native LDL, treatment with oxLDL (50-100 μM hydroperoxides) for 24 h significantly increased the levels of either cPLA₂ protein expression or constitutively phosphorylated cPLA₂ protein; in addition we observed enzyme translocation to membranes. iPLA₂ activity was not stimulated by oxLDL. Arachidonic acid release appeared to be associated with phosphorylation of ERK1/2 which was significantly enhanced in a dose-dependent manner whereas no activation of p38 and JNKs was found, indicating that these MAPKs are not involved in mediating the maximal oxLDL response. Western blotting on subcellular fractions and confocal microscopy analyses confirmed an increase in 15-lipoxygenase (15-LO) protein expression and translocation upon activation. A significant increase of cyclooxygenase-2 expression into membrane fraction was also found. Collectively, the data presented link the stimulation of ERK–cPLA₂–15-LO pathway by oxLDL to the prooxidant mechanism of the lipoprotein complex. It may initially stimulate the fibroblast reaction against the oxidation challenge as well as metabolic repair, such as during lung inflammation and pulmonary fibrosis.

Keywords: Phospholipase A; ₂; 15-lipoxygenase; Low-density lipoprotein; MAP kinases; Fibroblasts

Studies on the extra-mitochondrial CoA-ester formation of valproic and Δ⁴-valproic acids by Cátia C.P. Aires; Jos P.N. Ruiter; Paula B.M. Luís; Herman J. ten Brink; Lodewijk IJlst; Isabel Tavares de Almeida; Marinus Duran; Ronald J.A. Wanders; Margarida F.B. Silva (pp. 533-543).

The hypothesis whether valproic acid (VPA) and its main microsomal metabolite, Δ⁴-valproic acid, can be activated to the respective CoA esters in the cell cytosol was investigated. The valproyl-CoA formation was measured in different subcellular fractions obtained by differential centrifugation of liver homogenates of rats treated with VPA (studies ex vivo) and digitonin fractionation of rat hepatocytes incubated with VPA and cofactors (studies in vitro). The results show that VPA activation may occur in the cytosol and is not restricted to the mitochondrial matrix as believed until now. Furthermore, the activation of Δ⁴-VPA is demonstrated in vitro. Valproyl-CoA and Δ⁴-valproyl-CoA were detected after in vitro incubations and the former also in the mitochondrial and cytosolic fractions obtained from liver cells of treated rats. The activation to valproyl-CoA was characterized in cytosolic fractions, optimized with respect to time and protein and the kinetic constants ( K_m^app) were estimated for the reaction substrates. Other medium-chain fatty acids decreased the formation of valproyl-CoA suggesting a competition for both mitochondrial and extra-mitochondrial VPA activating enzymes. The present findings suggest additional mechanisms of mitochondrial dysfunction associated with VPA, and they may contribute to the further understanding of the toxic effects associated with this drug.

Keywords: Abbreviations; VPA; 2-; n; -propylpentanoic acid or valproic acid; Δ; ⁴; -VPA; 2-; n; -propyl-4-pentenoic acid; CoA; coenzyme A; dephCoA; dephosphocoenzyme A; P; i; orthophosphate; AMP; adenosine-5′-monophosphate; ADP; adenosine-5′-diphosphate; ATP; adenosine-5′-triphosphate; MES; 2-[N-morpholino]ethanesulfonic acid; MOPS; 3-[N-morpholino]propane-sulfonic acid; EGTA; ethylene glycol-bis(β-aminoethyl ether)-; N; ,; N; ,; N; ′,; N′; ,-tetraacetic acid; SEM buffer; S; ucrose/; E; GTA/; M; OPS buffer; BSA; bovine serum albumin; BCA; bicinchoninic acid; DTNB; 5,5′-dithio-bis(2-nitrobenzoic acid) or Ellman's reagent; ACS; acyl-CoA synthetase; RLM; rat liver mitochondria; HPLC; high performance liquid chromatography; FAO; fatty acid β-oxidation; MCFA; medium-chain fatty acids; LCFA; long-chain fatty acidsValproic acid; Δ; ⁴; -Valproic acid; Fatty acid activation; Acyl-CoA synthetase; Drug induced steatosis; Steatosis mechanism

Non-invasive analysis of gallbladder bile composition in cynomolgus monkeys using in vivo¹H magnetic resonance spectroscopy by Basil Künnecke; Andreas Bruns; Markus von Kienlin (pp. 544-549).

The purpose of the present study was (i) to establish a modality for non-invasively probing bile composition in cynomolgus monkeys and (ii) to ascertain the variability in biliary metabolism by repeatedly assessing gallbladder bile in situ. Localised in vivo¹H magnetic resonance spectroscopy (MRS) provided high-resolution spectra of gallbladder bile that allowed for the first time different species of bile acids, their taurine and glycine conjugates, and phospholipids to be identified and quantified in situ. A combined cross-sectional and longitudinal study of bile composition was conducted over 4 weeks in monkeys kept under standardised nutritional conditions. All biles were composed of the same major constituents. Bile acids contributed 267±47 μmol/ml whereof cholate, deoxycholate and chenodeoxycholate were the most abundant primary bile acids. Bile acid conjugation reached an extent of 100%. However, the actual quantitative contributions of different bile constituents varied distinctly. Correlation analysis revealed that intra-individual variability ( r=0.77±0.03) was significantly ( p<0.01) smaller than inter-individual variability ( r=0.68±0.01), thus purporting the notion that bile composition is a hallmark of individual metabolism. Extension of quantitative bile analysis by in vivo¹H-MRS to pathological states will provide a rapid and non-invasive modality for monitoring an important, yet elusive compartment of cholesterol and lipid metabolism.

Keywords: Abbreviations; AAALAC; Association for Assessment and Accreditation of Laboratory Animal Care International; MR; magnetic resonance; MRS; magnetic resonance spectroscopy; TE; echo time; TR; repetition timeMagnetic resonance spectroscopy; Bile acid; Cynomolgus monkey; Gallbladder; Bile conjugate

The liver plays a key role in whole body sterol accretion of the neonatal Golden Syrian hamster by Lihang Yao; Paul S. Horn; James E. Heubi; Laura A. Woollett (pp. 550-557).

Neonates have a significant requirement for cholesterol. From −1 to 25 days of age, the liver accrues 6.9 mg cholesterol and the extra-hepatic tissues accrue 107.7 mg cholesterol in the hamster. It is currently unknown if each of these body compartments synthesizes their own cholesterol or if they have alternative source(s) of sterol. Using³H₂O, in vivo hepatic sterol synthesis rates (per g liver per animal) increased between −1 and 5 days of age, decreased by 10 days of age, and increased again by 15 days of age. HMG-CoA reductase (HMGR) expression levels paralleled in vivo synthesis rates. Extra-hepatic sterol synthesis rates followed the same pattern as sterol synthesis rates in the liver. When sterol synthesis rates were converted to the mass of sterol synthesized per day, the liver synthesized 38.9 and the extra-hepatic tissues synthesized 63.9 mg cholesterol in the 26-day neonatal period. Comparing the amount of cholesterol accrued to that synthesized, one can conclude that the liver is a major source of sterol for the whole body during the neonatal period of the hamster. These results may help elucidate the cause(s) of reduced growth rates in neonates with liver disease or in neonates with compromised sterol synthesis rates.

Keywords: SLOS; Smith–Lemli–Opitz syndrome; DPS; digitonin-precipitable sterols; HMGR; HMG CoA Reductase; SREBP; sterol regulatory element binding proteinDevelopment; Cholesterol; Smith–Lemli–Opitz; Cholestasis; HMG-CoA reductase; Neonate

Polyunsaturated fatty acids stimulate phosphatidylcholine synthesis in PC12 cells by U. Ingrid Richardson; Richard J. Wurtman (pp. 558-563).

The synthesis of phospholipids in mammalian cells is regulated by the availability of three critical precursor pools: those of choline, cytidine triphosphate and diacylglycerol. Diacylglycerols containing polyunsaturated fatty acids (PUFAs) apparently are preferentially utilized for phosphatide synthesis. PUFAs are known to play an important role in the development and function of mammalian brains. We therefore studied the effects of unsaturated, monounsaturated and polyunsaturated fatty acids on the overall rates of phospholipid biosynthesis in PC12 rat pheochromocytoma cells. Docosahexaenoic acid (DHA, 22:6n-3), eicosapentaenoic acid (EPA, 20:5n-3) and arachidonic acid (AA, 20:4n-6) all significantly stimulated the incorporation of¹⁴C-choline into total cellular phospholipids. In contrast, monounsaturated oleic acid (OA) and the saturated palmitic (PA) and stearic (SA) acids did not have this effect. The action of DHA was concentration-dependent between 5 and 50 μM; it became statistically significant by 3 h after DHA treatment and then increased over the ensuing 3 h. DHA was preferentially incorporated into phosphatidylethanolamine (PE) and phosphatidylserine (PS), while AA predominated in phosphatidylcholine (PC).

Keywords: Membrane metabolism; Phospholipid; Docosahexaenoic acid; Eicosapentaenoic acid; Arachidonic acid; PC12 cell

Corrigendum to “Plasma cholesteryl ester transfer protein mass and phospholipid transfer protein activity are associated with leptin in type 2 diabetes mellitus” [Biochim. Biophys. Acta 1771 (2007) 113–118] by R.P.F. Dullaart; R. de Vries; G.M. Dallinga-Thie; A. van Tol; W.J. Sluiter (pp. 564-564).

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BBA - Molecular and Cell Biology of Lipids (v.1771, #4)