BBA - Molecular and Cell Biology of Lipids (v.1581, #3)
Retinal fatty acid binding protein reduce lipid peroxidation stimulated by long-chain fatty acid hydroperoxides on rod outer segments by Margarita H. Guajardo; Ana M. Terrasa; Angel Catalá (65-74).
In the present study we have investigated the effect of partially purified retinal fatty acid binding protein (FABP) against nonenzymatic lipid peroxidation stimulated by hydroperoxides derived from fatty acids on rod outer segment (ROS) membranes. Linoleic acid hydroperoxide (LHP), arachidonic acid hydroperoxide (AHP) and docosahexaenoic acid hydroperoxide (DHP) were prepared from linoleic acid, arachidonic acid and docosahexaenoic acid, respectively, by means of lipoxidase. ROS membranes were peroxidized using an ascorbate–Fe+2 experimental system. The effect on the peroxidation of ROS containing different amounts of lipid hydroperoxides (LOOH) was studied; ROS deprived of exogenously added LOOH was utilized as control. The degradative process was measured simultaneously by determining chemiluminescence and fatty acid composition of total lipids isolated from ROS. The addition of hydroperoxides to ROS produced a marked increase in light emission. This increase was hydroperoxide concentration-dependent. The highest value of activation was produced by DHP. The decrease percentage of the more polyunsaturated fatty acids (PUFAs) (20:4 n6 and 22:6 n3) was used to evaluate the fatty acid alterations observed during the process. We have compared the fatty acid composition of total lipids isolated from native ROS and peroxidized ROS that were incubated with and without hydroperoxides. The major difference in the fatty acid composition was found in the docosahexaenoic acid content, which decreased by 45.51±1.07% in the peroxidized group compared to native ROS; the decrease was even higher, 81.38±1.11%, when the lipid peroxidation was stimulated by DHP. Retinal FABP was partially purified from retinal cytosol. Afterwards, we measured its effect on the reaction of lipid peroxidation induced by LOOH. As a result, we observed a decrease of chemiluminescence (inhibition of lipid peroxidation) when adding increasing amounts (0.2 to 0.6 mg) of retinal FABP to ROS. The inhibitory effect reaches its highest value in the presence of DHP (41.81±10.18%). Under these conditions, bovine serum albumin (BSA) produces a smaller inhibitory effect (20.2±7.06%) than FABP.
Keywords: Retina; Rod outer segment; Lipid peroxidation; Fatty acid hydroperoxide; Fatty acid binding protein;
Secretory phospholipase A2 induces apoptosis via a mechanism involving ceramide generation by Sheng Zhao; Xiao-Yan Du; Min-Qiang Chai; Jun-Song Chen; Yuan-Chong Zhou; Jian-Guo Song (75-88).
Secretory phospholipase A2 (sPLA2) plays important roles in cellular signaling and various biological events. In this study, we examined the biological effects and the potential signaling mechanism of purified sPLA2 in MV1Lu cells. Three types of snake venom sPLA2 were purified and their enzymatic activities were characterized by using various lipid substrates prepared from [3H]-myristate-labeled cells and by determining their effects on the induction of arachidonic acid (AA) release. The purified sPLA2 induced apoptosis in Mv1Lu cells in a dose- and time-dependent manner, and was associated with a rapid increase in the intracellular ceramide level. Similar apoptotic effects were observed in Mv1Lu cells treated with exogenous ceramide analog, C2- and C8-ceramide. Moreover, treatment of cells with sphingomyelinase (SMase), which reduced the intracellular SM level, enhanced the apoptotic response to sPLA2s. sPLA2s also displayed an inhibitory effect on bradykinin-induced phospholipase D (PLD) activity, which can be imitated by exogenous ceramide. Our data indicate that sPLA2 induces cell apoptosis via a mechanism involving increased ceramide generation.
Keywords: Apoptosis; Ceramide; Phospholipase D; Secretory phospholipase A2; Sphingomyelinase; Sphingomyelin;
Conjugated linoleic acid decreases production of pro-inflammatory products in macrophages: evidence for a PPARγ-dependent mechanism by Y. Yu; P.H. Correll; J.P. Vanden Heuvel (89-99).
Conjugated linoleic acid (CLA) is a dietary fatty acid that has received considerable attention due to its unique properties in rodent models including anti-cancer, anti-atherogenic and anti-diabetic effects. The effects of CLA are similar to those seen with ligands for peroxisome proliferator-activated receptor (PPARs), most notably of the PPARγ subtype. With the recent observation of a role for PPARγ in regulation of immune responses, we suspected that CLA could affect immune function, in particular macrophage activity. The goal of our study was to examine whether this dietary fatty acid has anti-inflammatory properties similar to those reported for PPARγ activators such as 15-deoxy prostaglandin J2 (PGJ2). In reporter assays, various CLA isomers activated PPARγ in RAW264.7 mouse macrophage (RAW) cells. CLA decreased the interferon-γ (IFNγ)-induced mRNA expression of mediators of inflammation including cyclooxygenase 2 (COX2), inducible NOS (iNOS), and tumor necrosis factor α (TNFα). Reporter assays also demonstrated reduced IFNγ-stimulated transcriptional activity of the iNOS and COX2 promoters by CLA. Consequently, CLA decreased the production of PGE2, TNFα and the inflammatory agent nitric oxide (NO) in RAW cells treated with IFNγ. Other pro-inflammatory cytokines such as IL-1β and IL-6 were similarly decreased by CLA treatment of RAW cells. In addition, various CLA isomers induced HL60 cell differentiation along the monocytic lineage as assessed by measuring expression of the cell surface marker CD14. This differentiation process, as well as the regulation of iNOS and COX2 by 15dPGJ2, is believed to involve PPARγ. Mutations of Leu468 and Glu471 to alanine in helix 12 of the ligand-binding domain of PPARγ resulted in a protein with strong dominant-negative activity (dnPPARγ). Transfecting dnPPARγ into RAW cells eliminated the ability of various CLA isomers to regulate the iNOS reporter construct. Taken together, these results suggest that CLA has anti-inflammatory properties that are mediated, at least in part, by the nuclear hormone receptor PPARγ.
Keywords: Conjugated linoleic acid; Inducible nitric oxide synthetase; Cyclooxygenase; Peroxisome proliferator-activated receptor; Macrophage; Regulation of inflammation;
Protein-disulfide isomerase is a component of an NBD-cholesterol monomerizing protein complex from hamster small intestine by Tian-Quan Cai; Qiu Guo; Birming Wong; Denise Milot; Liwen Zhang; Samuel D Wright (100-108).
A rapid in vitro assay was developed for monitoring protein-mediated cholesterol monomerization from bile acid aggregates. This assay uses a fluorescent cholesterol analog, 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3β-ol (NBD-cholesterol), which was shown to be absorbed by hamster in a fashion similar to cholesterol. The fluorescence of aggregates of NBD-cholesterol was strongly quenched in 2.5 mM of taurocholic acid. Addition of proteins from enterocytes of hamster small intestine led to a time- and dose-dependent dequenching of NBD-cholesterol fluorescence. Comparable dequenching can be detected with SDS and appears to involve monomerization of the NBD-cholesterol. Purification of enterocyte extract by sequential chromatography revealed a ∼140-kDa protein complex (p140) able to mediate the monomerization of NBD-cholesterol. Each p140 complex mediated monomerization of 2.7 NBD-cholesterol molecules. The p140 complex appeared to be formed by dimerization of two ∼58-kDa molecules since SDS-PAGE revealed a single dominant band at 58 kDa (p58). Protein sequence analysis suggested that p58 is protein-disulfide isomerase (PDI), and this conclusion was confirmed by cloning of hamster PDI, and detection of PDI enzyme activity in the purified fraction. Additional studies with either pure PDI or lysates of cells transfected with hamster PDI showed that PDI by itself was not sufficient for monomerizing cholesterol. Further, despite a similar mobility on SDS-PAGE (∼58 kDa), the p140 complex appeared ∼45-kDa larger than pure PDI (∼95 kDa) when analyzed by a gel-filtration chromatography. The p140 complex may thus contain an unidentified molecule(s) in addition to PDI that may contribute importantly to cholesterol monomerization.
Keywords: Small intestine; Cholesterol monomerizing protein; Protein-disulfide isomerase;
Mutations in erg4 affect the sensitivity of Saccharomyces cerevisiae to medium-chain fatty acids by Virginia McDonough; Joseph Stukey; Trudy Cavanagh (109-118).
We have found that the medium-chain fatty acids (MCFAs) undecanoic acid (11:0), 10-undecenoic acid (11:1Δ10), and lauric acid (12:0) can affect the growth of Saccharomyces cerevisiae in a dose-dependent manner. The principal effect was a longer lag phase in MCFA-containing medium, although higher concentrations of 11:1Δ10 inhibited growth. Their relative order of inhibitory action was 11:1Δ10>11:0>12:0. Cellular content with MCFA supplementation was dependent on the concentration and the particular species of fatty acid, with 12:0 showing the highest relative accumulation and 11:1Δ10 the lowest at all concentrations. We have isolated and characterized a mutant that is hypersensitive to MCFA supplementation and is unable to grow at the normally permissive condition of 1 mM 11:1Δ10. However, it does not appear to accumulate higher relative levels of the fed MCFA compared to wild-type cells. Complementation of the mutant revealed that the ERG4 gene, encoding the enzyme that catalyzes the last step in ergosterol biosynthesis, had been mutated. The fatty acid composition of the erg4Δ mutant differs only slightly from wild-type cells, mainly involving an increase in the relative amount of 12:0. These results indicate that yeast require ergosterol for optimal growth on certain MCFAs. We discuss the role ergosterol may have in cells responding to exogenous MCFAs and in supporting optimal cell growth.
Keywords: ERG4; 10-Undecenoic acid; Medium-chain fatty acid; Ergosterol;
Paradoxical enhancement of hepatic metabolism of 7-ketocholesterol in sterol 27-hydroxylase-deficient mice by Malcolm A Lyons; Nobuyo Maeda; Andrew J Brown (119-126).
7-Ketocholesterol (7KC) is a major oxysterol found in atherosclerotic plaque and is believed to be derived both endogenously and exogenously (from the diet). Previously, we have demonstrated that subsequent to hepatic lipoprotein uptake, 7KC delivered in a model chylomicron remnant lipid emulsion is metabolised more rapidly and excreted into the intestinal tract and faeces to a much greater extent than simultaneously administered cholesterol. Furthermore, we have shown that human 7KC metabolism is dependent upon sterol 27-hydroxylase (27OHase). In the present work, we utilised a mouse model possessing the null mutation in the sterol 27-hydroxylase gene, Cyp27, to further investigate the metabolism and potential arterial accumulation of 7KC versus cholesterol. Despite the homozygous null mutation in Cyp27 (Cyp27−/−), 7KC was observed to undergo greater metabolism and excretion in the Cyp27−/− animals compared with the wild-type control mice. Six hours post-injection, 7KC levels were greater in aortae from Cyp27−/− mice but by 24 h, there was no significant difference between the knockout and control mice. We conclude that in contrast to humans, mice do not have an absolute requirement for 27OHase in order to metabolise 7KC and must rely on alternative side-chain oxidising pathways.
Keywords: 7-Oxocholesterol; Oxysterol; Cyp27; Diet; Mouse; Chylomicron remnant-like emulsion;
Distribution of microsomal triglyceride transfer protein within sub-endoplasmic reticulum regions in human hepatoma cells by Yusuke Higashi; Hiroyuki Itabe; Hironaga Fukase; Masahiro Mori; Yasuyuki Fujimoto; Ryuichiro Sato; Tsuneo Imanaka; Tatsuya Takano (127-136).
Very low-density lipoprotein (VLDL) particles are formed in the endoplasmic reticulum (ER) through the association of lipids with apolipoprotein B (apoB). Microsomal triglyceride transfer protein (MTP), which transfers lipid molecules to nascent apoB, is essential for VLDL formation in ER. However, little is known of the distribution and interaction of MTP with apoB within ER. In this study, distribution patterns of apoB and MTP large subunit (lMTP) within ER were examined. Microsomes prepared from HuH-7 cells, a human hepatoma cell line, were further fractionated into rough ER (RER)-enriched subfractions (ER-I fraction) and smooth ER (SER)-enriched subfractions (ER-II fraction) by iodixanol density-gradient ultracentrifugation. ApoB was evenly distributed in the ER-I and the ER-II fractions, while 1.5 times more lMTP molecules were present in the ER-I fraction than in the ER-II fraction. lMTP and apoB were coprecipitated both in the ER-I and in the ER-II fractions by immunoprecipitation whenever anti-apoB or an anti-lMTP antibodies were used. ApoB-containing lipoprotein particles showed a lower density in the ER-II fraction than those in the ER-I fraction. From these results, it is suggested that MTP can function in both rough and smooth regions of ER in human hepatoma cells.
Keywords: Apolipoprotein B; Endoplasmic reticulum; HuH-7; Lipid; Microsomal triglyceride transfer protein;