BBA - Molecular and Cell Biology of Lipids (v.1781, #10)

Fisetin, morin and myricetin attenuate CD36 expression and oxLDL uptake in U937-derived macrophages by Tzi-Wei Lian; Lisu Wang; Ya-Hsuan Lo; I-Jen Huang; Ming-Jiuan Wu (601-609).
Dietary flavonoid intake has been reported to be inversely associated with the incidence of coronary artery disease. To clarify the possible role of flavonoids in the prevention of atherosclerosis, we investigated the effects of some of these compounds, including fisetin, morin and myricetin, on the susceptibility of low-density lipoprotein (LDL) to oxidative modification and on oxLDL uptake in macrophages. The results demonstrated that fisetin had stronger inhibitory activity than the other two on inhibiting Cu2+-mediated LDL oxidation measured by thiobarbituric acid-reactive substances assay (TBARS), conjugated diene formation and electrophoretic mobility. The class B scavenger receptor, CD36, to which oxLDL binds, is present in atherosclerotic lesions. Treatment of U937-derived macrophages with myricetin (20 µM) significantly inhibited CD36 cell surface protein and mRNA expression (p  < 0.01). Fisetin, morin and myricetin (20 µM) also reduced the feed-forward induction of CD36 mRNA and surface protein expression by PPARγ. The inhibition of CD36 by flavonols was mediated by interference with PPARγ activation thus counteracting the deleterious autoamplification loop of CD36 expression stimulated by PPARγ ligand. All three flavonols (10 and 20 µM) markedly decreased the uptake of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanide perchlorate (DiI)-labeled oxLDL uptake in U937-derived macrophages dose-dependently. Current evidences indicate that fisetin, morin and myricetin not only prevent LDL from oxidation but also block oxLDL uptake by macrophages at least in part through reducing CD36 gene expression on macrophages. In conclusion, flavonols may play a role in ameliorating atherosclerosis.
Keywords: Atherogenesis; Fisetin; Morin; Myricetin; CD36; U937 cells; PPARγ;

The domain responsible for sphingomyelin synthase (SMS) activity by Calvin Yeang; Shweta Varshney; Renxiao Wang; Ya Zhang; Deyong Ye; Xian-Cheng Jiang (610-617).
Sphingomyelin synthase (SMS) sits at the crossroads of sphingomyelin (SM), ceramide, diacylglycerol (DAG) metabolism. It utilizes ceramide and phosphatidylcholine as substrates to produce SM and DAG, thereby regulating lipid messengers which play a role in cell survival and apoptosis. There are two isoforms of the enzyme, SMS1 and SMS2. Both SMS1 and SMS2 contain two histidines and one aspartic acid which are evolutionary conserved within the lipid phosphate phosphatase superfamily. In this study, we systematically mutated these amino acids using site-directed mutagenesis and found that each point mutation abolished SMS activity without altering cellular distribution. We also explored the domains which are responsible for cellular distribution of both enzymes. Given their role as a potential regulator of diseases, these findings, coupled with homology modeling of SMS1 and SMS2, will be useful for drug development targeting SMS.
Keywords: Sphingomyelin synthase 1 and 2; Point mutagenesis; Lipid phosphate phosphatase;

The contribution of triacylglycerol to energy provision in the hypertrophied heart, mediated through lipoprotein lipase (LPL) is largely unknown and the contribution of very-low-density lipoprotein (VLDL) receptor to control of LPL presentation at the endothelium is unclear. For isolated perfused rat hearts, cold acclimation (CA) induced volume-overload hypertrophy, with decreased developed pressure (P  < 0.01), increased end-diastolic volume of the left ventricle (P  < 0.001) and a loss of contractile reserve in response to dobutamine challenge (P  < 0.01). Oleate utilisation by perfused hearts was unchanged by CA, however uptake of intralipid emulsion increased 3-fold (P  < 0.01). CA increased the proportion of lipid deposited in tissue lipids from 10% in euthermic controls to 40% (P  < 0.01) although the overall contribution of individual lipid classes was unaffected. Cold acclimation significantly increased heparin-releasable LPL (P  < 0.05) and tissue residual LPL (P  < 0.01). Western blot analysis indicated preserved expression of proteins coding for SERCA2, muscle-CPT1 and VLDL-receptor following CA, while AMPKα2 and phospho-AMPKα2 were unaffected. These observations indicate that for physiological hypertrophy AMPK phosphorylation does not mediate the enhanced translocation of LPL to cardiac endothelium.
Keywords: Cardiac hypertrophy; Very-low-density lipoprotein receptor; AMPK; Triacylglycerol;

Rsp5p, a yeast S. cerevisiae ubiquitin ligase, is essential for regulation of unsaturated fatty acid synthesis via activation of the transcriptional activators Spt23p and Mga2p. Here we show that the conditional mutant rsp5-19 produces decreased levels of the end products of mevalonate pathway, such as ergosterol, ubiquinone and of dolichols, especially those with 19–24 isoprene units. The mechanism of Rsp5p involvement in the control of these lipid synthesis pathways was addressed by overproduction of Rsp5p-independent Spt23p or Mga2p. Expression of constitutively active forms of these transactivators resulted in excess production of ergosterol, but did not restore a wild-type level of dolichols. Moreover, synthesis of long chain dolichols was decreased in the wild-type and a rsp5-19 background. Finally, overproduction of active Spt23p or Mga2p was accompanied by the appearance of large lipid particles in the wild-type and rsp5-19 strains as observed by Nile Red staining, due to accumulation of unsaturated triacylglycerol. Thus, we conclude that Rsp5p, Spt23p and Mga2p may participate in the control of the homeostasis of lipids and lipid particles.
Keywords: Rsp5p ubiquitin ligase; Spt23p and Mga2p transcriptional activators; Sterols; Dolichols; Ubiquinone; Triacylglycerol;

Expression of Ca2+-independent and Ca2+-dependent phospholipases A2 and cyclooxygenases in human melanocytes and malignant melanoma cell lines by Mariagrazia Rita Scuderi; Carmelina Daniela Anfuso; Gabriella Lupo; Carla Motta; Loriana Romeo; Liliana Guerra; Alessandro Cappellani; Nicola Ragusa; Giuseppina Cantarella; Mario Alberghina (635-642).
We provide novel evidence that human melanoma cell lines (M10, M14, SK-MEL28, SK-MEL93, 243MEL, 1074MEL, OCM-1, and COLO38) expressed, at mRNA and protein levels, either Ca2+-independent phospholipase A2 (iPLA2) or cytosolic phospholipase A2 (cPLA2) and its phosphorylated form. Normal human melanocytes contained the lowest levels of both PLA2s. Cyclooxygenase-1 and -2 (COX-1 and COX-2) were also expressed in cultured tumor cells as measured by Western blots. The most pronounced overexpression of iPLA2 and COX-1 was found in two melanoma-derived cells, M14 and COLO38. Normal human melanocytes and the M10 melanoma cell line displayed no COX-2 expression. Using subcellular fractionation, Western blot and confocal microcopy analyses, in paradigmatic SK-MEL28 and SK-MEL93 cells we showed that iPLA2, COX-1 and even cPLA2 were equally located in the cytosol, membrane structures and perinuclear region while COX-2 was preferentially associated with the cytosol. Specific inhibitors of these three enzymes significantly reduced the basal proliferation rate either in melanocytes or in melanoma cell lines. These results, coupled with the inhibition of the cell proliferation by electroporation of melanoma cells with cPLA2 or COX-2 antibodies, demonstrate that a possible correlation between PLA2-COX expression and tumor cell proliferation in the melanocytic system does exist. In addition, the high expression level of both PLA2s and COXs suggests that eicosanoids modulate cell proliferation and tumor invasiveness.
Keywords: Phospholipase A2; Cyclooxygenase; Human melanoma; Melanocyte; Eicosanoid;

Inhibition of carboxylesterase 1 is associated with cholesteryl ester retention in human THP-1 monocyte/macrophages by J. Allen Crow; Brandy L. Middleton; Abdolsamad Borazjani; M. Jason Hatfield; Philip M. Potter; Matthew K. Ross (643-654).
Cholesteryl esters are hydrolyzed by cholesteryl ester hydrolase (CEH) yielding free cholesterol for export from macrophages. Hence, CEH has an important regulatory role in macrophage reverse cholesterol transport (RCT). CEH and human carboxylesterase 1 (CES1) appear to be the same enzyme. CES1 is inhibited by oxons, the bioactive metabolites of organophosphate (OP) pesticides. Here, we show that CES1 protein is robustly expressed in human THP-1 monocytes/macrophages and its biochemical activity inhibited following treatment of cell lysates and intact cells with chlorpyrifos oxon, paraoxon, or methyl paraoxon (with nanomolar IC50 values) or after immunodepletion of CES1 protein. CES1 protein expression in cells is unaffected by a 24-h paraoxon treatment, suggesting that the reduced hydrolytic activity is due to covalent inhibition of CES1 by oxons and not down-regulation of expression. Most significantly, treatment of cholesterol-loaded macrophages with either paraoxon (a non-specific CES inhibitor) or benzil (a specific CES inhibitor) caused enhanced retention of intracellular cholesteryl esters and a “foamy” phenotype, consistent with reduced cholesteryl ester mobilization. Thus, exposure to OP pesticides, which results in the inhibition of CES1, may also inhibit macrophage RCT, an important process in the regression of atherosclerosis.
Keywords: Atherosclerosis; Cholesteryl ester hydrolase; Carboxylesterase; Cholesterol metabolism; Organophosphate; Benzil;

Mass spectrometric analysis of lipid species of human circulating blood cells by Katharina Leidl; Gerhard Liebisch; Dorothea Richter; Gerd Schmitz (655-664).
Circulating blood cell lipid composition may become increasingly important to provide new insights into cellular lipid abnormalities in diseases. Here we compared lipid species in monocytes, lymphocytes, granulocytes, platelets and red blood cells (RBC) of healthy volunteers using electrospray ionization tandem mass spectrometry and detected striking differences among the examined blood cells. The different cell types were characterized by unique lipid class and lipid species pattern. The predominant lipid classes were phosphatidylcholine (PC) and free cholesterol (FC) with cell type specific PC/FC ratios as markers of membrane fluidity which was 1.9 in monocytes, 1.3 in lymphocytes, 1.1 in granulocytes, 0.8 in platelets and 0.3 in RBC, respectively. Beside a three-fold elevated ceramide level of 2.6 mol%, granulocytes revealed the highest percentage of phosphatidylethanolamine-based plasmalogens and a decreased fraction of highly polyunsaturated (≥ 3 double bonds) species compared to other cell types. Furthermore RBC showed a remarkable shift of glycerophospholipid chain length and platelets a nearly 4-fold increase of the cholesterol ester (CE) 18:2 (linoleic acid) fraction (55 mol% of total CE). In conclusion, the current study is a detailed comparison of lipid species in circulating blood cells of healthy human donors. This work could be a reference for studies in different patient cohorts directed towards discovery of novel lipid biomarkers in circulating blood cells.
Keywords: Monocytes; Lymphocytes; Granulocytes; Platelets; Red blood cell; ESI-MS/MS; Phospholipid; Lipidomics;