BBA - Molecular and Cell Biology of Lipids (v.1734, #3)

Contents (CO4).

N-Acylethanolamines (NAEs) and N-acylphosphatidylethanolamines (NAPEs) are trace constituents of vertebrate cells and tissues and much is known about their metabolism and possible function in animals. Here we report for the first time the identification and quantification of NAEs and NAPEs in several strains of the yeast Saccharomyces cerevisiae. Gas chromatography-mass spectrometry of appropriate derivatives revealed 16:0, 16:1, 18:0 and 18:1 N-acyl groups in both NAE and NAPE whose levels, in wild-type cells, were 50 to 90 and 85 to 750 pmol/μmol lipid P, respectively (depending on the phase of growth). NAPE levels were reduced by 45 to 60% in a strain lacking three type B phospholipases, suggesting their involvement in NAPE synthesis by their known transacylation activity. A yeast strain lacking the YPL103c gene, which codes for a protein with 50.3% homology to human NAPE-specific phospholipase D, exhibited a 60% reduction in NAE, compared to wild-type controls. The exposure of various yeast strains to peroxidative stress, by incubation in media containing 0.6 mM H2O2, resulted in substantial increases in NAE. Because yeast cells lack polyunsaturated fatty acids, they offer a useful system for the study of NAE generation and its potential signaling and cytoprotective effects in the absence of polyunsaturated (“endocannabinoid”) congeners.
Keywords: N-Acylethanolamine; N-Acylphosphatidylethanolamine; Yeast (Saccharomyces cerevisiae); Mutant strains; Oxidative stress;

Molecular mechanisms and regulation of ceramide transport by Ryan J. Perry; Neale D. Ridgway (220-234).
De novo biosynthesis of sphingolipids begins in the endoplasmic reticulum (ER) and continues in the Golgi apparatus and plasma membrane. A crucial step in sphingolipid biosynthesis is the transport of ceramide by vesicular and non-vesicular mechanisms from its site of synthesis in the ER to the Golgi apparatus. The recent discovery of the ceramide transport protein CERT has revealed a novel pathway for the delivery of ceramide to the Golgi apparatus for sphingomyelin (SM) synthesis. In addition to a ceramide-binding START domain, CERT has FFAT (referring to two phenylalanines [FF] in an acidic tract) and pleckstrin homology (PH) domains that recognize the ER integral membrane protein VAMP-associated protein (VAP) and Golgi-associated PtdIns 4-phosphate, respectively. Mechanisms for vectorial transport involving dual-organellar targeting and sites of deposition of ceramide in the Golgi apparatus are proposed. Similar Golgi–ER targeting motifs are also present in the oxysterol-binding protein (OSBP), which regulates ceramide transport and SM synthesis in an oxysterol-dependent manner. Consequently, this emerges as a potential mechanism for integration of sphingolipid and cholesterol metabolism. The identification of organellar targeting motifs in other related lipid-binding/transport proteins indicate that concepts learned from the study of ceramide transport can be applied to other lipid transport processes.
Keywords: Ceramide; Hydroxycholesterol; Lipid transport; CERT; Endoplasmic reticulum; Golgi apparatus;

Down-regulated expression of PPARα target genes, reduced fatty acid oxidation and altered fatty acid composition in the liver of mice transgenic for hTNFα by Heidi Glosli; Oddrun Anita Gudbrandsen; Antony J. Mullen; Bente Halvorsen; Therese H. Røst; Hege Wergedahl; Hans Prydz; Pål Aukrust; Rolf K. Berge (235-246).
The present study investigated the hepatic regulation of fatty acid metabolism in hTNFα transgenic mice. Reduced hepatic mRNA levels and activities of carnitine palmitoyltransferase-II (CPT-II) and mitochondrial HMG-CoA synthase were observed, accompanied by decreased fatty acid oxidation, fatty acyl-CoA oxidase and fatty acid synthase (FAS) activities and down-regulated gene expression of mitochondrial acetyl-CoA carboxylase 2 (ACC2). The mRNA levels of peroxisome proliferator-activated receptor α (PPARα) and PPARδ were reduced. The hepatic fatty acid composition was altered, with increased amounts of saturated and polyunsaturated fatty acids. The relative amounts of Δ9 desaturated fatty acids were decreased, as was Δ9desaturase mRNA. The CPT-I mRNA level remained unchanged. The PPARα targeted genes CPT-II and HMG-CoA synthase are potential regulators of mitochondrial fatty acid oxidation and ketogenesis in hTNFα transgenic mice, and the increased propionyl-CoA level found is a possible inhibitor of these processes. Reduced mitochondrial and peroxisomal fatty acid oxidation may explain the increased hepatic triglyceride level induced by TNFα. This is not due to de novo fatty acid synthesis as both FAS activity and gene expression of ACC2 were reduced.
Keywords: Mitochondrial and peroxisomal β-oxidation; Carnitine palmitoyltransferase; Desaturation; Fatty acid synthase; Fatty acyl-CoA oxidase; HMG-CoA synthase;

Profiling candidate genes involved in wax biosynthesis in Arabidopsis thaliana by microarray analysis by Patricia Costaglioli; Jérôme Joubès; Christel Garcia; Marianne Stef; Benoît Arveiler; René Lessire; Bertrand Garbay (247-258).
Plant epidermal wax forms a hydrophobic layer covering aerial plant organs which constitutes a barrier against uncontrolled water loss and biotic stresses. Wax biosynthesis requires the coordinated activity of a large number of enzymes for the formation of saturated very-long-chain fatty acids and their further transformation in several aliphatic compounds. We found in the available database 282 candidate genes that may play a role in wax synthesis, regulation and transport. To identify the most interesting candidates, we measured the level of expression of 204 genes in the aerial parts of 15-day-old Arabidopsis seedlings by performing microarray experiments. We showed that only 25% of the putative candidates were expressed to significant levels in our samples, thus significantly reducing the number of genes which will be worth studying using reverse genetics to demonstrate their involvement in wax accumulation. We identified a beta-keto acyl-CoA synthase gene, At5g43760, which is co-regulated with the wax gene CER6 in a number of conditions and organs. By contrast, we showed that neither the fatty acyl-CoA reductase genes nor the wax synthase genes were expressed in 15-day-old leaves and stems, raising questions about the identity of the enzymes involved in the acyl-reduction pathway that accounts for 20% of the total wax amount.
Keywords: Microarray; Wax synthesis; Arabidopsis thaliana;

PPARα-deficiency in mice fed a high-carbohydrate, low-cholesterol diet was associated with a decreased weight of epididymal adipose tissue and an increased concentration of adipose tissue cholesterol. Consumption of a high (2% w/w) cholesterol diet resulted in a further increase in the concentration of cholesterol and a further decrease in epididymal fat pad weight in PPARα-null mice, but had no effect in the wild-type. These reductions in fat pad weight were associated with an increase in hepatic triacylglycerol content, indicating that both PPARα-deficiency and cholesterol altered the distribution of triacylglycerol in the body. Adipose tissue de novo lipogenesis was increased in PPARα-null mice and was further enhanced when they were fed a cholesterol-rich diet; no such effect was observed in the wild-type mice. The increased lipogenesis in the chow-fed PPARα-null mice was accompanied paradoxically by lower mRNA expression of SREBP-1c and its target genes, acetyl-CoA carboxylase and fatty acid synthase. Consumption of a high-cholesterol diet increased the mRNA expression of these genes in the PPARα-deficient mice but not in the wild-type. De novo cholesterol synthesis was not detectable in the adipose tissue of either genotype despite a relatively high expression of the mRNA's encoding SREBP-2 and 3-hydroxy-3-methylglutaryl Coenzyme A reductase. The mRNA expression of these genes and of the LDL-receptor in adipose tissue of the PPARα-deficient mice was lower than that of the wild-type and was not downregulated by cholesterol feeding. The results suggest that PPARα plays a role in adipose tissue cholesterol and triacylglycerol homeostasis and prevents cholesterol-mediated changes in de novo lipogenesis.
Keywords: Adipose tissue; PPARα; Cholesterol; Lipogenic flux; mRNA expression; SREBP;

Effect of conjugated linoleic acid isomers on lipoproteins and atherosclerosis in the Syrian Golden hamster by Patricia L. Mitchell; Morgan A. Langille; Deborah L. Currie; Roger S. McLeod (269-276).
Conjugated linoleic acid (CLA) is a group of positional and geometric isomers of linoleic acid (LA, C18:2 cis-9, cis-12) that are reported to have important biological activities, including protection against atherosclerosis. In this study, the potential role of the individual cis-9, trans-11 and trans-10, cis-12 isomers of CLA in atherogenesis were compared with LA in the Syrian Golden hamster. Supplementation of a high-fat, high-cholesterol diet (HFHC) with 1% (w/w) cis-9, trans-11 CLA or trans-10, cis-12 CLA did not significantly affect plasma cholesterol levels compared to supplementation with 1% (w/w) LA. Very low density lipoprotein cholesterol (VLDL-C) was lower and plasma triglycerides (TG) were higher in diets where C18:2 fatty acid was added to the HFHC diet, but neither the cis-9, trans-11 CLA group nor trans-10, cis-12 CLA group was significantly different from the LA control group. CLA supplementation did not significantly affect low density lipoprotein cholesterol (LDL-C). Trans-10, cis-12 CLA increased high density lipoprotein cholesterol (HDL-C) levels compared to LA or cis-9, trans-11 CLA (P  < 0.02), and although the ratio of non-HDL-C:HDL-C in the cis-9, trans-11 CLA group (1.11 ± 0.54) and the trans-10, cis-12 CLA group (1.11 ± 0.21) was lower than the LA group (1.29 ± 0.45), the reduction did not reach statistical significance. Atherosclerosis was assessed in the ascending aorta by measuring the number of aortic cross-sections containing Oil Red O-stained intimal lesions. Compared to the LA group (60 ± 11%), both the cis-9, trans-11 CLA group (38 ± 8%) and the trans-10, cis-12 CLA group (28 ± 7%) had fewer sections displaying a fatty streak lesion, although the differences did not reach statistical significance. These results suggest that individual CLA isomers may reduce atherosclerotic lesion development in the hamster, but when compared to LA, the apparent atheroprotective effects do not correlate with beneficial changes in lipoprotein profile.
Keywords: Conjugated linoleic acid; Atherosclerosis; Lipoprotein; Hamster;

Fatty acid metabolism in Atlantic salmon (Salmo salar L.) hepatocytes and influence of dietary vegetable oil by Ingunn Stubhaug; Douglas R. Tocher; J. Gordon Bell; James R. Dick; Bente E. Torstensen (277-288).
Isolated hepatocytes from Atlantic salmon (Salmo salar), fed diets containing either 100% fish oil or a vegetable oil blend replacing 75% of the fish oil, were incubated with a range of seven 14C-labelled fatty acids. The fatty acids were [1-14C]16:0, [1-14C]18:1n-9, 91-14C]18:2n-6, [1-14C]18:3n-3, [1-14C]20:4n-6, [1-14C]20:5n-3, and [1-14C]22:6n-3. After 2 h of incubation, the hepatocytes and medium were analysed for acid soluble products, incorporation into lipid classes, and hepatocytes for desaturation and elongation. Uptake into hepatocytes was highest with [1-14C]18:2n-6 and [1-14C]20:5n-3 and lowest with [1-14C]16:0. The highest recovery of radioactivity in the cells was found in triacylglycerols. Of the phospholipids, the highest recovery was found in phosphatidylcholine, with [1-14C]16:0 and [1-14C]22:6n-3 being the most prominent fatty acids. The rates of β-oxidation were as follows: 20:4n-6 > 18:2n-6 = 16:0 > 18:1n-9 > 22:6n-3 = 18:3n-3 = 20:5n-3. Of the fatty acids taken up by the hepatocytes, [1-14C]16:0 and [1-14C]18:1n-9 were subsequently exported the most, with the majority of radioactivity recovered in phospholipids and triacylglycerols, respectively. The major products from desaturation and elongation were generally one cycle of elongation of the fatty acids. Diet had a clear effect on the overall lipid metabolism, with replacing 75% of the fish oil with vegetable oil resulting in decreased uptake of all fatty acids and reduced incorporation of fatty acids into cellular lipids, but increased β-oxidation activity and higher recovery in products of desaturation and elongation of [1-14C]18:2n-6 and [1-14C]18:3n-3.
Keywords: Uptake; Phospholipid; TAG; β-Oxidation; Secretion; Salmon;

Cumulative Contents (289-290).