BBA - Molecular and Cell Biology of Lipids (v.1761, #3)

Alkaline sphingomyelinase (alk-SMase) is present in the intestinal tract and additionally human bile. It hydrolyses sphingomyelin in both intestinal lumen and the mucosal membrane in a specific bile salt dependent manner. The enzyme was discovered 36 years ago but got real attention only in the last decade, when sphingomyelin metabolism was realized to be a source of multiple lipid messengers, and when dietary sphingomyelin was found to inhibit colonic tumorigenesis in animals. The enzyme shares no structural similarity with other SMases and belongs to the nucleotide pyrophosphatase/phosphodiesterase family. The enzyme is of specific properties, such as bile salt dependency, trypsin resistance, high stability, and tissue specific expression. In the colon, the enzyme may play antiproliferative and antiinflammatory roles through generating ceramide, reducing the formation of lysophosphatidic acid, and inactivating platelet-activating factor. The enzyme is down regulated in human long-standing ulcerative colitis and colonic adenocarcinoma, and mutation of the enzyme has been found in colon cancer cells. In the small intestine, alk-SMase is the key enzyme for sphingomyelin digestion. The hydrolysis of sphingomyelin may affect the cholesterol uptake and have impact on sphingomyelin levels in plasma lipoproteins. The review summarizes the new information of alk-SMase from biochemical, cell and molecular biological studies in the last decade and evaluates its potential implications in development of colon cancer, inflammatory bowel diseases, and atherosclerosis.
Keywords: Alkaline sphingomyelinase; Colon cancer; Inflammatory bowel diseases; Sphingomyelin digestion; Atherosclerosis; Platelet activating factor; Lysophosphatidic acid; Nucleotide pyrophosphatase phosphodiesterase; Intestine; Liver;

Elevated plasma levels of high-density lipoprotein cholesterol (HDL-C) are atheroprotective and HDL-dependent reverse cholesterol transport has been related to this effect. HDL particles may, however, undergo modifications that affect their biological activities. Lipoxygenases (LOs) belong to a family of lipid peroxidizing enzymes; among them, reticulocyte-type 15-lipoxygenase (15-LO-1) appears to play a pathophysiological role in atherosclerosis, as its expression is increased in atherosclerotic plaques and it has been shown to oxidize low-density lipoproteins to an atherogenic form. In this work we investigated the impact of in vitro 15-lipoxygenase-catalyzed modification of HDL3 on their ability to act as cholesterol acceptor and found that 15-LO-modified HDL3 were less effective in mediating cholesterol efflux from lipid-laden J774 cells. A reduced binding of 15-LO-modified HDL3 to scavenger receptor class B, type I (SR-BI), due to HDL apoproteins cross-linking, explained, at least in part, the observed reduction of cholesterol efflux. In addition, ATP-binding cassette transporter A1 (ABCA1)-mediated cholesterol efflux was also reduced, as a consequence of pre-β-particles loss after HDL3 modification. These results suggest that 15-lipoxygenase might induce structural alterations of HDL3 particles that impair their capability of triggering reverse cholesterol transport.
Keywords: High-density lipoprotein; 15-lipoxygenase; Reverse cholesterol transport; Scavenger receptor class B, type I; ATP-binding cassette transporter A1;

Biosynthesis of C9-aldehydes in the moss Physcomitrella patens by Michael Stumpe; Julia Bode; Cornelia Göbel; Thomas Wichard; Andreas Schaaf; Wolfgang Frank; Markus Frank; Ralf Reski; Georg Pohnert; Ivo Feussner (301-312).
After wounding, the moss Physcomitrella patens emits fatty acid derived volatiles like octenal, octenols and (2E)-nonenal. Flowering plants produce nonenal from C18-fatty acids via lipoxygenase and hydroperoxide lyase reactions, but the moss exploits the C20 precursor arachidonic acid for the formation of these oxylipins. We describe the isolation of the first cDNA (PpHPL) encoding a hydroperoxide lyase from a lower eukaryotic organism. The physiological pathway allocation and characterization of a downstream enal-isomerase gives a new picture for the formation of fatty acid derived volatiles from lower plants. Expression of a fusion protein with a yellow fluorescent protein in moss protoplasts showed that PpHPL was found in clusters in membranes of plastids. PpHPL can be classified as an unspecific hydroperoxide lyase having a substrate preference for 9-hydroperoxides of C18-fatty acids but also the predominant substrate 12-hydroperoxy arachidonic acid is accepted. Feeding experiments using arachidonic acid show an increase in the 12-hydroperoxide being metabolized to C8-aldehydes/alcohols and (3Z)-nonenal, which is rapidly isomerized to (2E)-nonenal. PpHPL knock out lines failed to emit (2E)-nonenal while formation of C8-volatiles was not affected indicating that in contrast to flowering plants, PpHPL is only involved in formation of a specific subset of volatiles.
Keywords: Nonenal; Cytochrome P-450; Lipid peroxidation; Oxylipin metabolism; Moss; Substrate specificity;

Phospholipid homeostasis in phosphatidylserine synthase-2-deficient mice by Rineke Steenbergen; Terry S. Nanowski; Randy Nelson; Stephen G. Young; Jean E. Vance (313-323).
Phosphatidylserine (PS) is synthesized in mammalian cells by two distinct serine-exchange enzymes, phosphatidylserine synthase-1 and -2. We recently demonstrated that mice lacking PS synthase-2 develop normally and exhibit no overt abnormalities [Bergo et al., (2002) J. Biol. Chem. 277:47701–47708]. We now show that PS synthase-2 mRNA levels are up to 80-fold higher in livers of embryos than in adults. Despite reduced serine-exchange activity in several tissues of PS synthase-2 deficient mice, the phospholipid composition of mitochondria and microsomes from these tissues is normal. Although PS synthase-2 is highly expressed in neurons, axon extension of cultured sympathetic neurons is not impaired by PS synthase-2 deficiency. We hypothesized that mice compensate for PS synthase-2 deficiency by modifying their phospholipid metabolism. Our data show that the rate of PS synthesis in hepatocytes is not reduced by PS synthase-2 deficiency but PS synthase-1 activity is increased. Moreover, PS degradation is decreased by PS synthase-2 deficiency, probably as a result of decreased PS degradation via phospholipases rather than decreased PS decarboxylation. These experiments underscore the idea that cellular phospholipid composition is tightly controlled and show that PS synthase-2-deficient hepatocytes modify phospholipid metabolism by several compensatory mechanisms to maintain phospholipid homeostasis.
Keywords: Phosphatidylserine; Phosphatidylethanolamine; Phosphatidylserine synthase; Phosphatidylserine decarboxylase; Hepatocytes; Knock-out mice;

Plant fatty acid (ethanol) amide hydrolases by Rhidaya Shrestha; Sang-chul Kim; John M. Dyer; Richard A. Dixon; Kent D. Chapman (324-334).
Fatty acid amide hydrolase (FAAH) plays a central role in modulating endogenous N-acylethanolamine (NAE) levels in vertebrates, and, in part, constitutes an “endocannabinoid” signaling pathway that regulates diverse physiological and behavioral processes in animals. Recently, an Arabidopsis FAAH homologue was identified which catalyzed the hydrolysis of NAEs in vitro suggesting a FAAH-mediated pathway exists in plants for the metabolism of endogenous NAEs. Here, we provide evidence to support this concept by identifying candidate FAAH genes in monocots (Oryza sativa) and legumes (Medicago truncatula), which have similar, but not identical, exon–intron organizations. Corresponding M. truncatula and rice cDNAs were isolated and cloned into prokaryotic expression vectors and expressed as recombinant proteins in Escherichia coli. NAE amidohydrolase assays confirmed that these proteins indeed catalyzed the hydrolysis of 14C-labeled NAEs in vitro. Kinetic parameters and inhibition properties of the rice FAAH were similar to those of Arabidopsis and rat FAAH, but not identical. Sequence alignments and motif analysis of plant FAAH enzymes revealed a conserved domain organization for these members of the amidase superfamily. Five amino-acid residues determined to be important for catalysis by rat FAAH were absolutely conserved within the FAAH sequences of six plant species. Homology modeling of the plant FAAH proteins using the rat FAAH crystal structure as a template revealed a conserved protein core that formed the active site of each enzyme. Collectively, these results indicate that plant and mammalian FAAH proteins have similar structure/activity relationships despite limited overall sequence identity. Defining the molecular properties of NAE amidohydrolase enzymes in plants will help to better understand the metabolic regulation of NAE lipid mediators.
Keywords: Lipid metabolism; N-acylethanolamines; Fatty acid amide hydrolase;

Distribution of cholesterol and galactosylceramide in rat cerebellar white matter by Katrin Börner; Håkan Nygren; Birgit Hagenhoff; Per Malmberg; Elke Tallarek; Jan-Eric Månsson (335-344).
White matter and the inner granular layer of rat cerebellum was analysed by imaging time-of-flight secondary-ion mass spectrometry (TOF-SIMS) equipped with a Bi+ ion cluster gun. Samples were prepared by high pressure freezing, freeze-fracturing and freeze drying or by plunge freezing and cryostat sectioning. The identified and localized chemical species were: sodium, potassium, phosphocholine, cholesterol and galactosylceramide (GalC) with carbon chain lengths C18:0 (N-stearoyl-galactosylceramide) and C24:0 (N-lignoceroylgalactosylceramide) with CH24:0 (hydroxy-lignoceroylgalactosylceramide).We report new findings regarding the organization of myelin in white matter. One is cholesterol-rich, ribbon-shaped 10–20 μm areas excluding Na+ and K+. The second finding is the different distribution of GalC C18 and GalC C24 in relation to these areas, where GalC C18 was localized in cholesterol-rich areas and GalC C24 was localized in Na/K-enriched areas. The distribution of GalC was in small spots, homogeneous in size, of 0.8–1.5 μm. Sample preparation with high pressure freezing allowed separate localization of sodium and potassium in tissue samples.
Keywords: Cholesterol; Galactosylceramide; Phosphocholine; Sodium; Potassium; Localization; Cerebellum;

The effects of structured triglycerides containing one long chain fatty acid (oleic acid, C18:1) and one short chain saturated fatty acid (caprylic acid, 8:0) on lipidemia, liver and aortic cholesterol, and fecal neutral sterol excretion were investigated in male Golden Syrian hamsters fed a hypercholesterolemic regimen consisting of 89.9% commercial ration to which was added 10% coconut oil and 0.1% cholesterol (w/w). After 2 weeks on the HCD diet, the hamsters were bled, following an overnight fast (16 h) and placed into one of three dietary treatments of eight animals each based on similar plasma cholesterol levels. The hamsters either continued on the HCD diet or were placed on diets in which the coconut oil was replaced by one of two structured triglycerides, namely, 1(3),2-dicaproyl-3(1)-oleoylglycerol (OCC) or 1,3-dicaproyl-2-oleoylglycerol (COC) at 10% by weight. Plasma total cholesterol (TC) in hamsters fed the OCC and COC compared to the HCD were reduced 40% and 49%, respectively (P  < 0.05). Similarly, hamsters fed the OCC and COC diets reduced their plasma nonHDL cholesterol levels by 47% and 57%, respectively (P  < 0.05), compared to hamsters fed the HCD after 2 weeks of dietary treatment. Although hamsters fed the OCC (−26%) and COC (−32%) had significantly lower plasma HDL levels compared to HCD, (P  < 0.05), the plasma nonHDL/HDL cholesterol ratio was significantly lower (P  < 0.05) compared to the HCD for the OCC-fed (−27%) and the COC-fed (−38%) hamsters, respectively. Compared to the HCD group, aortic esterified cholesterol was 20% and 53% lower for the OCC and COC groups, respectively, with the latter reaching statistical significance, P  < 0.05. In conclusion, the hamsters fed the structured triglyceride oils had lower blood cholesterol levels and lower aortic accumulation of cholesterol compared to the control fed hamsters.
Keywords: Structured triglycerides; Oleic acid; Plasma cholesterol; Aortic cholesterol; Hamsters;

The protective effect of ebselen, with documented glutathione peroxidase-like activity and antioxidative and anti-inflammatory properties, on the cytotoxicity induced by oxysterol was investigated in ECV-304 cells with cholestane-3β, 5α, 6β-triol (3-triol), one of the most toxic oxysterols. 3-triol exhibited significant cytotoxicity to ECV-304 cells in dose- and time-dependent manners. Pre-incubations with ebselen at different concentrations for 4 h effectively inhibited the decreases of the cell viability and the intracellular thiols level induced by 3-triol; suppressed the 3-triol-caused increases of the GPx and NOS activities, the LDH leakage and MDA formation. The inhibition of ebselen to the generation of intracellular ROS induced by 3-triol was monitored by luminol-, lucigenin-derived chemiluminescence and DCFH-DA-derived fluorescence assays. Our results suggest that ebselen inhibited 3-triol-induced enhancement of intracellular ROS level and the cytotoxicity of 3-triol is contributed to, for the most part, an enhanced formation of intracellular O2 ·−; nevertheless, the mitochondria were not the main source of intercellular O2 ·− contributed to the cytotoxicity of 3-triol. Ebselen lost its high protection against 3-triol-induced injuries in the presence of GSH probably due to the formation of the ebselen-GSH adduct. In conclusion, our investigations provide new utility for ebselen as a prospective antiatherosclerotic in both clinical and non-clinical situations.
Keywords: Antiatherosclerotic; Ebselen; Oxysterol; Oxidative stress; Reactive oxygen species;

The fungus Aspergillus tamarii transforms progesterone 1 into testololactone 5 in high yield through a four-step enzymatic pathway which is flexible to a range of steroidal substrates. To date, no studies have investigated the fate of C-6 (ring-B) and C-11 (ring-C) functionalized steroidal substrates on metabolism. Remarkably all of the C-6 functionalized substrates underwent reductive metabolism on ring-A in contrast to C-11 functionalized steroids where only ring-D oxidative or reductive transformation occurred. In order to discern the precise role of the functional groups in directing metabolism 6-ketoprogesterone 10 with functionality at C-6 and the ring-D methyl ketone underwent reductive and oxidative transformation on both terminal A and D rings showing that this functionality was directing metabolism. Androst-4-en-3,6-dione 12 devoid of ring-D functionality underwent reductive metabolism on ring-A proving that the C-6 functionality was directing metabolism to this ring with the ring-D methyl ketone responsible for generating transformation at this position. Functionality at C-11 exclusively controlled entry into and degree of metabolism on the lactonization pathway. These novel findings may have important bearing in the future understanding of structure activity relationships in revealing new metabolic pathways and further affords a unique opportunity for generation of novel bioactive steroidal compounds.
Keywords: Steroid transformation; Aspergillus tamarii; Baeyer–Villiger oxidation; Androst-4-en-3-one; Pregn-4-en-3-one;

Histamine increases sphingosine kinase-1 expression and activity in the human arterial endothelial cell line EA.hy 926 by a PKC-α-dependent mechanism by Andrea Huwiler; Frauke Döll; Shuyu Ren; Sabine Klawitter; Anna Greening; Isolde Römer; Svetlana Bubnova; Luise Reinsberg; Josef Pfeilschifter (367-376).
Sphingosine 1-phosphate (S1P) is a potent mitogenic signal generated from sphingosine by the action of sphingosine kinases (SKs). In this study, we show that in the human arterial endothelial cell line EA.hy 926 histamine induces a time-dependent upregulation of the SK-1 mRNA and protein expression which is followed by increased SK-1 activity. A similar upregulation of SK-1 is also observed with the direct protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA). In contrast, SK-2 activity is not affected by neither histamine nor TPA. The increased SK-1 protein expression is due to stimulated de novo synthesis since cycloheximide inhibited the delayed SK-1 protein upregulation. Moreover, the increased SK-1 mRNA expression results from an increased promoter activation by histamine and TPA. In mechanistic terms, the transcriptional upregulation of SK-1 is dependent on PKC and the extracellular signal-regulated protein kinase (ERK) cascade since staurosporine and the MEK inhibitor U0126 abolish the TPA-induced SK-1 induction. Furthermore, the histamine effect is abolished by the H1-receptor antagonist diphenhydramine, but not by the H2-receptor antagonist cimetidine. Parallel to the induction of SK-1, histamine and TPA stimulate an increased migration of endothelial cells, which is prevented by depletion of the SK-1 by small interfering RNA (siRNA). To appoint this specific cell response to a specific PKC isoenzyme, siRNA of PKC-α, -δ, and -ε were used to selectively downregulate the respective isoforms. Interestingly, only depletion of PKC-α leads to a complete loss of TPA- and histamine-triggered SK-1 induction and cell migration. In summary, these data show that PKC-α activation in endothelial cells by histamine-activated H1-receptors, or by direct PKC activators leads to a sustained upregulation of the SK-1 protein expression and activity which, in turn, is critically involved in the mechanism of endothelial cell migration.
Keywords: Histamine; Phorbol ester; PKC isoenzyme; Sphingosine kinase-1; Endothelial cell; Migration;

Formation of conjugated linoleic acid metabolites in human vascular endothelial cells by Robert Ringseis; André Müller; Kaja Düsterloh; Sabine Schleser; Klaus Eder; Hans Steinhart (377-383).
Conjugated linoleic acids (CLAs) are bioactive lipid compounds showing anti-atherogenic actions in cell culture experiments and animal models of atherosclerosis without exact knowledge about the underlying mechanisms. CLAs were recently reported to be further metabolized to bioactive conjugated metabolites indicating that these metabolites are possibly involved in mediating the anti-atherogenic actions of CLA. Regarding the lack of information with respect to the formation of CLA metabolites in the vascular endothelium, which is strongly involved in the process of atherosclerosis, the present study aimed to explore the potential formation of CLA metabolites in vascular endothelial cells. The results from the present study show for the first time that the CLA isomers cis-9, trans-11 CLA and trans-10, cis-12 CLA are metabolized within endothelial cells to β-oxidation products such as CD16:2c7t9 and CD16:2t8c10 and elongation products such as CD20:2c11t13, CD20:2t12c14 as well as CD22:2c13t15 and CD22:2t14c16. Different CD16:2/CLA ratios observed between cells treated with different CLA isomers indicate that the metabolism of CLAs depends on the configuration of the conjugated double bonds. In conclusion, regarding the biological activity reported for CD20:2t12c14 and other metabolites of CLA, the present results indicate that metabolites of CLA are possibly also involved in mediating the anti-atherogenic actions of CLA.
Keywords: Conjugated linoleic acid; Atherosclerosis; Human aortic endothelial cell; Conjugated dienoic structure; cis-9, trans-11 CLA; trans-10, cis-12 CLA;