BBA - Molecular and Cell Biology of Lipids (v.1761, #4)

Oxidative stress and arachidonic acid mobilization by María A. Balboa; Jesús Balsinde (385-391).
Reactive oxygen species are known to contribute to tissue damage during injury and inflammation. However, these species can also be sensed by the cells and trigger intracellular signaling cascades. This review examines recent evidence on the involvement of reactive oxygen species in lipid signaling. Attention is focused on activation of phospholipase A2s, enzymes whose action on membrane phospholipids can also render molecules with opposite effects on cells. The participation of Ca2+-dependent and Ca2+-independent phospholipase A2s in arachidonic acid mobilization from phospholipids is discussed, with particular attention to the interplay between cytosolic and secreted Ca2+-dependent forms. The involvement of alternative routes for arachidonic acid mobilization under oxidative stress is also considered.
Keywords: Oxidative stress; Arachidonic acid; Lysophosphatidylcholine; Phospholipase A2; Eicosanoids;

Modification of low-density lipoprotein by myeloperoxidase-derived oxidants and reagent hypochlorous acid by Ernst Malle; Gunther Marsche; Jürgen Arnhold; Michael J. Davies (392-415).
Substantial evidence supports the notion that oxidative processes contribute to the pathogenesis of atherosclerosis and coronary heart disease. The nature of the oxidants that give rise to the elevated levels of oxidised lipids and proteins, and decreased levels of antioxidants, detected in human atherosclerotic lesions are, however, unclear, with multiple species having been invoked. Over the last few years, considerable data have been obtained in support of the hypothesis that oxidants generated by the heme enzyme myeloperoxidase play a key role in oxidation reactions in the artery wall. In this article, the evidence for a role of myeloperoxidase, and oxidants generated therefrom, in the modification of low-density lipoprotein, the major source of lipids in atherosclerotic lesions, is reviewed. Particular emphasis is placed on the reactions of the reactive species generated by this enzyme, the mechanisms and sites of damage, the role of modification of the different components of low-density lipoprotein, and the biological consequences of such oxidation on cell types present in the artery wall and in the circulation, respectively.
Keywords: MPO-H2O2-chloride system; Hypochlorite; 2-chlorohexadecanal; Thiocyanate; Atherosclerosis; Glomerulosclerosis; Lipid oxidation; Protein oxidation; Radicals;

Several lines of evidence suggest that lipid rafts are involved in cellular fatty acid uptake and influence fatty acid translocase (FAT/CD36) function. However, it remains unknown whether caveolae, a specialized raft type, are required for this mechanism. Here, we show that wild-type (WT) mouse embryonic fibroblasts (MEFs) and caveolin-1 knockout (KO) MEFs, which are devoid of caveolae, have comparable overall expression of FAT/CD36 protein but altered subcellular FAT/CD36 localization and function. In WT MEFs, FAT/CD36 was isolated with both lipid raft enriched detergent-resistant membranes (DRMs) and detergent-soluble membranes (DSMs), whereas in cav-1 KO cells it was exclusively associated with DSMs. Subcellular fractionation demonstrated that FAT/CD36 in WT MEFs was localized intracellularly and at the plasma membrane level while in cav-1 KO MEFs it was absent from the plasma membrane. This mistargeting of FAT/CD36 in cav-1 KO cells resulted in reduced fatty acid uptake compared to WT controls. Adenoviral expression of caveolin-1 in KO MEFs induced caveolae formation, redirection of FAT/CD36 to the plasma membrane and rescue of fatty acid uptake. In conclusion, our data provide evidence that caveolin-1 is necessary to target FAT/CD36 to the plasma membrane. Caveolin-1 may influence fatty acid uptake by regulating surface availability of FAT/CD36.
Keywords: Lipid raft; Caveolin-1; Caveolae; Fatty acid translocase;

Serum amyloid A (SAA)-induced remodeling of CSF-HDL by Takashi Miida; Toshiyuki Yamada; Utako Seino; Masayuki Ito; Yuriko Fueki; Akihiro Takahashi; Keiichiro Kosuge; Satoshi Soda; Osamu Hanyu; Konen Obayashi; Osamu Miyazaki; Masahiko Okada (424-433).
Inflammation is a risk factor for Alzheimer's disease. Serum amyloid A (SAA) is an acute phase protein that dissociates apolipoprotein AI (apoAI) from plasma HDL. In cerebrospinal fluid (CSF), the SAA concentration is much higher in subjects with Alzheimer's disease than in controls. CSF-HDL is rich in apoE, which plays an important role as a ligand for lipoprotein receptors in the central nervous system (CNS). To clarify whether SAA dissociates apoE from CSF-HDL, we added recombinant SAA to CSF and determined the apoE distribution in the CSF using native two-dimensional gel electrophoresis. We found that SAA dissociated apoE from CSF-HDL in a dose-dependent manner. This effect was more evident in apoE4 carriers than in apoE3 or apoE2 carriers. After a 24-h incubation at 37 °C, SAA continuously dissociated apoE from CSF-HDL. Amyloid β (Aβ) fragments (1–42) were bound to large CSF-HDL but not to apoE dissociated by SAA. In conclusion, SAA dissociates apoE from CSF-HDL. We postulate that inflammation in the CNS may impair Aβ clearance due to the loss of apoE from CSF-HDL.
Keywords: Alzheimer's disease; SAA; Apolipoprotein E; amyloid β; Inflammation; Cerebrospinal fluid;

Several types of prostaglandin (PG)s are synthesized in adipocytes and involved differently in the control of adipogenesis. To elucidate how the PG synthesis is regulated at different stages in the life cycle of adipocytes, we examined the gene expression of arachidonate cyclooxygenase (COX) pathway leading to the delayed synthesis of PGE2 and PGF and their roles in adipogenesis after exposure of cultured cells to phorbol 12-myristate 13-acetate (PMA), which is a useful system for monitoring mitogen-induced changes. While the expression of COX-1 remained constitutive, mRNA and protein levels of COX-2 were up-regulated by treatment with PMA. Preadipocytes exhibited higher gene expression of cytosolic phospholipase A2α (cPLA2α) and PGF synthase. In contrast, three isoforms of PGE synthase are expressed constitutively during all phases. The delayed synthesis of PGE2 and PGF following the stimulation for 24 with a mixture of PMA and calcium ionophore A23187 was the highest in preadipocytes, reflecting the increased expression levels of cPLA2α and COX-2. Cultured cells treated with PMA during the differentiation phase and then exposed to the maturation medium, or cells treated with PMA in the maturation medium after the differentiation phase showed the suppression of adipogenesis in adipocytes. The attenuating effect of PMA was additionally enhanced when the cell were treated along with A32187 during the differentiation phase, suggesting the involvement of endogenous PGs. The cells at the stages of the differentiation and maturation phases were highly sensitive to exogenous PGE2 and PGF, respectively, resulting in the marked suppression of the stored fats in adipocytes. Taken together, these results provided the evidence for the distinct gene expression of isoformic enzymes in the COX pathway leading to the synthesis of PGE2 and PGF and the specific action of these prostanoids at different cycle stages of adipocytes.
Keywords: Cyclooxygenase; Prostanoid; Adipocyte; Phorbol 12-myristate 13-acetate; A23187, 3T3-L1 cell;

Dietary plant sterols accumulate in the brain by Paula J. Jansen; Dieter Lütjohann; Karlygash Abildayeva; Tim Vanmierlo; Torsten Plösch; Jogchum Plat; Klaus von Bergmann; Albert K. Groen; Frans C.S. Ramaekers; Folkert Kuipers; Monique Mulder (445-453).
Dietary plant sterols and cholesterol have a comparable chemical structure. It is generally assumed that cholesterol and plant sterols do not cross the blood–brain barrier, but quantitative data are lacking. Here, we report that mice deficient for ATP-binding cassette transporter G5 (Abcg5) or Abcg8, with strongly elevated serum plant sterol levels, display dramatically increased (7- to 16-fold) plant sterol levels in the brain. Apolipoprotein E (ApoE)-deficient mice also displayed elevated serum plant sterol levels, which was however not associated with significant changes in brain plant sterol levels. Abcg5- and Abcg8-deficient mice were found to carry circulating plant sterols predominantly in high-density lipoprotein (HDL)-particles, whereas ApoE-deficient mice accommodated most of their serum plant sterols in very low-density lipoprotein (VLDL)-particles. This suggests an important role for HDL and/or ApoE in the transfer of plant sterols into the brain. Moreover, sitosterol upregulated apoE mRNA and protein levels in astrocytoma, but not in neuroblastoma cells, to a higher extend than cholesterol. In conclusion, dietary plant sterols pass the blood–brain barrier and accumulate in the brain, where they may exert brain cell type-specific effects.
Keywords: Cholesterol; Plant sterol; Campesterol; Sitosterol; Apolipoprotein E; ABCG5; ABCG8;

We previously reported that docosahexaenoic acid (DHA) attenuated tumor necrosis factor (TNF)-induced apoptosis in human monocytic U937 cells (J. Nutr. 130: 1095–1101, 2000). In the present study, we examined the effects of DHA and other polyunsaturated fatty acids (PUFA) on TNF-induced necrosis, another mode of cell death, using L929 murine fibrosarcoma cells. After preincubation with PUFA conjugated with BSA for 24 h, cells were treated with TNF or TNF + actinomycin D (Act D). Preincubation of cells with DHA enriched this polyunsaturated acid in the phospholipids and attenuated cell death induced by either TNF or TNF + Act D. When cells were treated with TNF alone, DNA laddering was not detected, and cells were coincidently stained with both annexin V-FITC and propidium iodide, indicating that the death mode was necrotic. TNF + Act D predominantly induced necrosis, although concurrent apoptotic cell death was also observed in this case. Preincubation with oleic acid, linoleic acid or 20:3(n-3) did not affect TNF-induced necrosis. Conversely, supplementation with n-3 docosapentaenoic acid (DPAn-3) or eicosapentaenoic acid (EPA) reduced necrotic cell death, but to a lesser extent in comparison with DHA. Unlike the case of U937 cell apoptosis, arachidonic acid (AA) significantly attenuated L929 cell necrosis, and 20:3(n-6) or 22:4(n-6) showed similar or less activity, respectively. Statistical evaluation indicated that the order of effective PUFA activity was DHA > DPAn-3 ≥ EPA > AA ≈ 20:3(n-6) ≥ 22:4(n-6). One step desaturation, C2 elongation or C2 cleavage within the n-6 or n-3 fatty acid group was probably very active in L929 cells, because AA, synthesized from 20:3(n-6) or 22:4(n-6), and C22 fatty acids, synthesized from AA or EPA, were preferentially retained in cellular phospholipids. These observations suggested that attenuation of TNF-induced necrosis by the supplementation of various C20 or C22 polyunsaturated fatty acids is mainly attributable to the enrichment of three kinds of polyunsaturated fatty acids, i.e., DHA, DPAn-3 or AA, in phospholipids. Among these fatty acids, DHA was the most effective in the reduction of L929 necrosis as observed in the case of U937 apoptosis. This suggests that DHA-enriched membranes can protect cell against TNF irrespective of death modes and that membranous DHA may abrogate the death signaling common to necrosis and apoptosis.
Keywords: Docosahexaenoic acid; Polyunsaturated fatty acid; Tumor necrosis factor; Necrosis; L929 cell;

Attenuated secretion of very low density lipoproteins from McA-RH7777 cells treated with eicosapentaenoic acid is associated with impaired utilization of triacylglycerol synthesized via phospholipid remodeling by Khai Tran; Fengcheng Sun; Zheng Cui; Gro Thorne-Tjomsland; Carly St. Germain; Louis R. Lapierre; Roger S. McLeod; James C. Jamieson; Zemin Yao (463-473).
In McA-RH7777 cells stably expressing human apolipoprotein (apo) B100, treatment with oleic acid (18:1(n-9)) promoted whereas treatment with eicosapentaenoic acid (EPA, 20:5(n-3)) attenuated assembly and secretion of VLDL. Under conditions where the cells were cultured in the presence of 20% serum, EPA (0.4 mM) had marginal effect on the secretion of total apoB100 (determined by pulse-chase analysis) but decreased (by 50%) secretion of triacylglycerol (TG), indicating that the inhibitory effect of EPA was exerted primarily on TG-rich VLDL. Analysis of phospholipid mass and species by tandem mass spectrometry showed increased phosphatidylethanolamine (PE) in EPA-treated cells, the increase was significant in the distal Golgi membranes (by 170%) and endoplasmic reticulum (by 116%). Lipid pulse-chase studies showed a major distinction between phospholipid species containing 20:5(n-3) and 18:1(n-9), which in turn was associated with distinct compartmentalization of TG containing 20:5(n-3) or 18:1(n-9) between cytosol and microsomes and their recruitment during VLDL assembly. Thus, 18:1-TG was secreted as VLDL but 20:5-TG was not. These results suggest that EPA attenuation of VLDL secretion is associated with impaired utilization of TG derived from phospholipid remodeling.
Keywords: Eicosapentaenoic acid; VLDL; Phospholipid; Triacylglycerol; McA-RH7777 cell;

Increasing evidence indicates that DHHC cysteine-rich domain-containing proteins (DHHC proteins) are protein acyltransferases. Although multiple DHHC proteins are found in eukaryotes, characterization has been examined for only a few. Here, we have cloned all the yeast and human DHHC genes and investigated their intracellular localization and tissue-specific expression. Most DHHC proteins are localized in the ER and/or Golgi, with a few localized in the plasma membrane and one in the yeast vacuole. Human DHHC mRNAs also differ in their tissue-specific expression. These results may provide clues to aid in discovering the specific function(s) of each DHHC protein.
Keywords: DHHC cysteine-rich domain; Palmitoylation; Protein acyltransferase;

Hypothyroidism down-regulates mitochondrial citrate carrier activity and expression in rat liver by Anna M. Giudetti; Monica Leo; Luisa Siculella; Gabriele V. Gnoni (484-491).
The effect of hypothyroidism on citrate carrier (CiC) activity has been investigated in rat-liver mitochondria. The rate of citrate transport was reduced by ∼50% in mitochondria from hypothyroid as compared with euthyroid rats. In parallel, a decrease in the rate of de novo fatty acid synthesis was observed in the cytosol of the former animals. Kinetic analysis of citrate transport revealed that only the V max was reduced by hypothyroidism, while K m was almost unaffected. Hypothyroidism increased the mitochondrial percentage of phosphatidylcholine while decreased that of phosphatidylethanolamine; an altered fatty acid pattern but no significant difference in the sum of saturated and unsaturated fatty acids as well as in the unsaturation index was observed. The CiC Arrhenius plot did not show appreciable difference between the two groups of rats. However, Western blot analysis associated with mRNA quantitation indicated that both protein level and mRNA accumulation of hepatic CiC were noticeably decreased in hypothyroid state. Therefore, a reduced content of the carrier protein can represent a plausible mechanism to explain the decline in the CiC activity observed in rat liver mitochondria of hypothyroid rats.
Keywords: Citrate carrier activity and expression; Fatty acids; Hypothyroidism; Lipogenesis; Mitochondria; Phospholipid;

Characterization of the rat LDL receptor 5′-flanking region by Dayami Lopez; Gene C. Ness (492-500).
A 1.5-kb genomic DNA fragment corresponding to the 5′-flanking region of the rat LDL receptor gene was cloned and putative regulatory regions were identified. A major transcription start site was identified at −154 bp relative to the ATG translation initiation codon, within a region containing two thyroid hormone response element half-site motifs (2H-TRE). Binding of thyroid hormone receptors α and β1 to this element was demonstrated. Mutations within this 2H-TRE region abolished basal transcription levels of the rat LDL receptor gene. Reporter gene studies indicated that the promoter region between −300 and −200 bp, which contains one sterol response element (SRE) and two specificity protein-1 sites (Sp1) sites, is crucial for basal transcription of the rat LDL receptor gene. The functionality of the SRE motif was confirmed using electrophoretic mobility shift assays and reporter gene studies.
Keywords: Promoter; Response element; Transcription start site; Rapid amplification of cDNA end; Thyroid hormone; SREBP;