BBA - Molecular and Cell Biology of Lipids (v.1781, #6-7)

Adaptation to oxidative stress induced by polyunsaturated fatty acids in yeast by Ana Cipak; Morana Jaganjac; Oksana Tehlivets; Sepp D. Kohlwein; Neven Zarkovic (283-287).
To create a conditional system for molecular analysis of effects of polyunsaturated fatty acids (PUFA) on cellular physiology, we have constructed a strain of yeast (Saccharomyces cerevisiae) that functionally expresses, under defined conditions, the Δ12 desaturase gene from the tropical rubber tree, Hevea brasiliensis. This strain produces up to 15% PUFA, exclusively under inducing conditions resulting in production of 4-hydroxy-2-nonenal, one of the major end products of n  − 6 polyunsaturated fatty acid peroxidation. The PUFA-producing yeast was initially more sensitive to oxidative stress than the wild-type strain. However, over extended time of cultivation it became more resistant to hydrogen peroxide indicating adaptation to endogenous oxidative stress caused by the presence of PUFA. Indeed, PUFA-producing strain showed an increased concentration of endogenous ROS, while initially increased hydrogen peroxide sensitivity was followed by an increase in catalase activity and adaptation to oxidative stress. The deletion mutants constructed to be defective in the catalase activity lost the ability to adapt to oxidative stress.These data demonstrate that the cellular synthesis of PUFA induces endogenous oxidative stress which is overcome by cellular adaptation based on the catalase activity.
Keywords: Fatty acid peroxidation; Reactive oxygen species; Saccharomyces cerevisiae; Fatty acid desaturase; Catalase;

Human lens lipids differ markedly from those of commonly used experimental animals by Jane M. Deeley; Todd W. Mitchell; Xiaojia Wei; John Korth; Jessica R. Nealon; Stephen J. Blanksby; Roger J.W. Truscott (288-298).
Electrospray ionisation tandem mass spectrometry has allowed the unambiguous identification and quantification of individual lens phospholipids in human and six animal models. Using this approach ca. 100 unique phospholipids have been characterised. Parallel analysis of the same lens extracts by a novel direct-insertion electron-ionization technique found the cholesterol content of human lenses to be significantly higher (ca. 6 times) than lenses from the other animals.The most abundant phospholipids in all the lenses examined were choline-containing phospholipids. In rat, mouse, sheep, cow, pig and chicken, these were present largely as phosphatidylcholines, in contrast 66% of the total phospholipid in Homo sapiens was sphingomyelin, with the most abundant being dihydrosphingomyelins, in particular SM(d18:0/16:0) and SM(d18:0/24:1). The abundant glycerophospholipids within human lenses were found to be predominantly phosphatidylethanolamines and phosphatidylserines with surprisingly high concentrations of ether-linked alkyl chains identified in both classes. This study is the first to identify the phospholipid class (head-group) and assign the constituent fatty acid(s) for each lipid molecule and to quantify individual lens phospholipids using internal standards. These data clearly indicate marked differences in the membrane lipid composition of the human lens compared to commonly used animal models and thus predict a significant variation in the membrane properties of human lens fibre cells compared to those of other animals.
Keywords: Cataract; Mass spectrometry; Electrospray ionisation; Fatty acid; Phospholipid; Cholesterol; Ether lipid;

We have previously demonstrated that exogenous nitric oxide (NO) directly inhibits alveolar macrophage (AM) cell-free activity of the enzyme 5-lipoxygenase (5-LO), thereby inhibiting metabolism of arachidonic acid to the important proinflammatory lipid mediators, leukotrienes (LT). Here, we explored the possibility that NO indirectly inhibited AM LT synthesis via activation of soluble guanylyl cyclase (sGC) in rat AM. The selective sGC inhibitor, LY83583, abrogated the suppression of cellular LT synthesis elicited by either exogenous or endogenous NO. A non-NO-dependent activator of sGC, YC-1, also inhibited macrophage LT synthesis. We next determined if sGC-mediated suppression of AM LT synthesis was dependent on protein kinase G (cGK). The selective cGK inhibitor, KT5823, reversed the suppression of cellular 5-LO metabolism following treatment with exogenous NO and YC-1. cGK1 activation resulted in phosphorylation of 5-LO. In contrast to peritoneal macrophages, AM exhibited localization of sGC, cGK1 and cGKII to the cell nucleus. In summary, in addition to its direct effects, NO-induced suppression of 5-LO action can be mediated indirectly through activation of the sGC and cGK pathways in AM. The nuclear localization of enzymes sGC, CGK1 and cGKII in the AM, which also demonstrates preferential nuclear 5-LO expression, may confer tighter regulation of LT synthesis.
Keywords: Endotoxin; Leukotrienes; Nitric oxide; cGMP; Protein kinase G;

The ATP-binding cassette transporters ABCA1 and ABCG1 as well as scavenger receptor BI (SR-BI) mediate the efflux of lipids from macrophages to apolipoprotein A-I (apoA-I) and high density lipoproteins (HDL). We used RNA interference in RAW264.7 macrophages to study the interactions of ABCA1, ABCG1, and SR-BI with lipid-free apoA-I, native and reconstituted HDL with apoA-I:phosphatidylcholine ratios of either 1:40 (rHDL1:40) or 1:100 (rHDL1:100). Knock-down of ABCA1 inhibits the cellular binding at 4 °C of lipid-free apoA-I but not of HDL whereas suppression of ABCG1 or SR-BI reduces the binding of HDL but not lipid-free apoA-I. The degree of lipidation influences the interactions of rHDL with ABCG1 and SR-BI. Knock-down of ABCG1 inhibits more effectively the binding and cholesterol efflux capacities of lipid-poorer rHDL1:40 whereas knock-down of SR-BI has a more profound effect on the binding and cholesterol efflux capacities of lipid-richer rHDL1:100. Moreover, knock-down of ABCG1 but not SR-BI interferes with the association of lipid-free apoA-I during prolonged incubation at 37 °C. Finally, knock-down of ABCG1 inhibits the binding of initially lipid-free apoA-I which has been preconditioned by cells with high ABCA1 activity. The gained ability of initially lipid-free apoA-I to interact with ABCG1 is accompanied by its shift from electrophoretic pre-β- to α-mobility. Taken together, these data suggest that the interaction of lipid-free apoA-I with ABCA1 generates a particle that immediately interacts with ABCG1 but not with SR-BI. Furthermore, the degree of lipidation influences the interaction of HDL with ABCG1 or SR-BI.
Keywords: ABC1; ABCG1; SR-BI; Apolipoprotein A-I; High density lipoproteins; Cholesterol efflux; Macrophages;

Activation of PPARγ reverses a defect of surfactant synthesis in mice lacking two types of fatty acid binding protein by Christian Schachtrup; Stefan Malcharek; Jack J. Haitsma; Burkhard Lachmann; Yuji Owada; Bert Binas; Hisatake Kondo; Bernd Rüstow; Hans-Joachim Galla; Friedrich Spener (314-320).
Lung surfactant is a lipid–protein-film covering the inner alveolar surface. We have previously shown that double knock-out (d-ko) mice lacking both the epidermal-type (E-) and the heart-type (H-) fatty acid binding protein (FABP) exhibit a defect of surfactant synthesis in alveolar type II cells that can be corrected by feeding pioglitazone, a drug that activates peroxisome proliferator-activated receptor gamma (PPARγ). Here, we demonstrate first that healthy surfactant at collapse pressure produces protrusions composed of bilayers but not folds, second that the d-ko effect profoundly perturbs lipid/hydrophobic protein composition, pressure-area isotherm, and structural organisation of the surfactant at nanoscale, parameters that are critical for the normal breathing cycle. In support of these data in vivo measurements of lung function reveal that maximum compliance in d-ko vs. wild-type mice is significantly reduced. Further, we show that the biophysical phenotype can be corrected substantially with pioglitazone. Finally, we show that d-ko alveolar cells up-regulate liver-type (L-) FABP, a member of the FABP family that we have previously shown to interact with PPARγ. Taken together, these data suggest that PPARγ agonists could be a tool to repair surfactant damage caused by dysfunctional alveolar lipid metabolism, and provide in vivo support for L-FABP aided signaling.
Keywords: Phospholipid synthesis; Fatty acid binding protein; Fatty acid signaling; Surfactant organisation; Lung compliance;

Metformin reduces cellular lysophosphatidylcholine and thereby may lower apolipoprotein B secretion in primary human hepatocytes by Josef Wanninger; Markus Neumeier; Johanna Weigert; Gerhard Liebisch; Thomas S. Weiss; Andreas Schäffler; Charalampos Aslanidis; Gerd Schmitz; Jürgen Schölmerich; Christa Buechler (321-325).
The biguanide metformin is an oral antihyperglycemic drug for the treatment of type 2 diabetes mellitus. Further, a moderate improvement of dyslipidemia by metformin was reported, and therefore, the effect of metformin on the release of apolipoprotein B (ApoB) and ApoE in primary human hepatocytes was determined. Metformin at 0.5 and 1 mM reduced hepatic ApoB secretion but ApoE was not altered. Metformin is well known to stimulate the AMP kinase that subsequently reduces hepatic nuclear factor 4-α (HNF4-α) and HNF4-α regulated genes like ApoB. However, HNF4-α was only diminished by 1 mM metformin and ApoB mRNA was not suppressed indicating that this pathway may not explain reduced ApoB release. Lower abundance of lysophosphatidylcholine (lysoPC) may also diminish ApoB secretion. Therefore, electrospray ionization tandem mass spectrometry was applied to measure cellular lipids. PC, lysoPC (produced by hydrolysis of PC), phosphatidylserine and sphingomyelin (derived from PC) were lower in metformin-treated hepatocytes whereas phosphatidylethanolamine, an alternative precursor of PC, was not affected. In addition, ABCB4, the canalicular membrane flippase essential for biliary PC secretion, was diminished. Supplementation with lysoPC led to a selective elevation of endogenous lysoPC and rescued ApoB secretion in metformin-treated cells. Therefore, it is concluded that metformin reduces lysoPC in human hepatocytes and this may secondarily lead to a therapeutically beneficial lower release of ApoB.
Keywords: Dyslipidemia; Apolipoprotein B; Metformin; Phosphatidylcholine; ABCB4;

Changes in fatty acids metabolism during differentiation of Atlantic salmon preadipocytes; Effects of n-3 and n-9 fatty acids by Marijana Todorčević; Anne Vegusdal; Tor Gjøen; Hilde Sundvold; Bente E. Torstensen; Marte A. Kjær; Bente Ruyter (326-335).
Atlantic salmon (Salmo salar) preadipocytes, isolated from visceral adipose tissue, differentiate from an unspecialized fibroblast like cell type to mature adipocytes filled with lipid droplets in culture. The expression of the adipogenic gene markers peroxisome proliferated activated receptor (PPAR) α, lipoprotein lipase (LPL), microsomal triglyceride transfer protein (MTP), fatty acid transport protein (FATP) 1 and fatty acid binding protein (FABP) 3 increased during differentiation. In addition, we describe a novel alternatively spliced form of PPARγ (PPARγ short), the expression of which increased during differentiation. Eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA) lowered the triacylglycerol (TAG) accumulation in mature salmon adipocytes compared to oleic acid (18:1n-9, OA). This finding indicates that a reduced level of highly unsaturated n-3 fatty acids (HUFAs) in fish diets, when the traditional marine oil is exchanged for n-9 fatty acids (FAs) rich vegetable oils (VOs), may influence visceral fat deposition in salmonids. Moreover, major differences in the metabolism of EPA, DHA and OA at different stages during differentiation of adipocytes occur. Most of the EPA and DHA were oxidized in preadipocytes, while they were mainly stored in TAGs in mature adipocytes in contrast to OA which was primarily stored in TAGs at all stages of differentiation.
Keywords: Atlantic salmon; Preadipocytes; Adipocytes; Differentiation; Lipid metabolism;

Modification of LDL with oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (oxPAPC) results in a novel form of minimally modified LDL that modulates gene expression in macrophages by Mathijs Groeneweg; Monique N. Vergouwe; Peter G. Scheffer; Hendrikus P.A. Vermue; Maarten D. Sollewijn Gelpke; Anneke M. Sijbers; Norbert Leitinger; Marten H. Hofker; Menno P.J. de Winther (336-343).
Oxidized phospholipids (oxPL) have been found in atherosclerotic plaques and have been associated with the development of atherosclerosis. To investigate the LDL modifying effects of oxPL and subsequent consequences for macrophages, murine bone marrow macrophages were treated with LDL modified with the oxidation products of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine, oxPAPC. Effects of oxPAPC-modified LDL (oxPAPC-LDL) on macrophages were compared to native LDL, copper oxidized LDL (Cu-oxLDL) and acetylated LDL (acLDL). LDL treatment with oxPAPC or CuSO4 induced active oxidation of the LDL particles, resulting in significant increase in F2-isoprostanes, typical for LDL oxidation, compared with native LDL, acLDL or PAPC-LDL. Uptake of oxPAPC-LDL in macrophages was mediated by CD36 pathways as shown by SR-A and CD36 inhibitors. Surprisingly, both Cu-oxLDL and acLDL induced cholesterol ester accumulation in macrophages while oxPAPC-LDL did not. Microarray analysis showed a pronounced similarity between Cu-oxLDL and oxPAPC-LDL gene expression induction, whereas acLDL and oxPAPC-LDL did not overlap. The main feature shared by oxPAPC-LDL and Cu-oxLDL was the induction of the glutathione dependent antioxidant response, indicating an important role in protecting against both types of modified LDL induced stress. Our experiments show that oxPL modifies LDL, resulting in a minimally oxidized particle that shares many features of classically copper oxidized LDL and that may have relevant in vivo properties.
Keywords: Atherogenesis; Foam cell; Oxidized phospholipid; LDL;

Molecular probing of the Saccharomyces cerevisiae sterol 24-C methyltransferase reveals multiple amino acid residues involved with C2-transfer activity by Kulothungan Ganapathy; Christopher W. Jones; Camille M. Stephens; Rit Vatsyayan; Julie A. Marshall; W. David Nes (344-351).
Two families of sterol C24-methyltransferase (SMT) are responsible for the formation of the ergostane (C1-transfer activity; SMT1) and stigmastane (C2-transfer activity: SMT2) sterol side chains, respectively. The fungal Saccharomyces cerevisiae SMT1 (Erg6p) operates the first C1-transfer in concerted fashion to form a single product whereas the protozoan and plant SMTs are bifunctional capable of catalyzing two sequential, mechanistically distinct C-methylation activities in the conversion of a Δ24-sterol acceptor to diverse doubly alkylated products. Previous mutation of the amino acids of Erg6p at D79, Y81 and E82 afforded C1 or C2-transfer activities typical of the protozoan and plant SMT. In this study, scanning mutagenesis experiments involving a leucine replacement of 52 amino acids in Erg6p followed by substitution of key residues with functionally or structurally similar amino acids indicated that 5 new residues at positions Y192, G217, G218, T219 and Y223 can switch the course of C1-transfer activity to include plant-like C2-transfer activity. The data support a model in which several conserved and non-conserved amino acids located in distinct regions of the Erg6p regulate the course of the C-methylation reaction toward product differences.
Keywords: Zymosterol; Fecosterol; Site-directed mutagenesis; Sterol catalysis; Sterol methyltransferase yeast; Saccharomyces cerevisiae; Active site; Erg6p;

Mice deficient in mitochondrial glycerol-3-phosphate acyltransferase-1 have diminished myocardial triacylglycerol accumulation during lipogenic diet and altered phospholipid fatty acid composition by Tal M. Lewin; Hendrik de Jong; Nicole J.M. Schwerbrock; Linda E. Hammond; Steven M. Watkins; Terry P. Combs; Rosalind A. Coleman (352-358).
Glycerol-3-phosphate acyltransferase-1 (GPAT1), which is located on the outer mitochondrial membrane comprises up to 30% of total GPAT activity in the heart. It is one of at least four mammalian GPAT isoforms known to catalyze the initial, committed, and rate-limiting step of glycerolipid synthesis. Because excess triacylglycerol (TAG) accumulates in cardiomyocytes in obesity and type 2 diabetes, we determined whether lack of GPAT1 would alter the synthesis of heart TAG and phospholipids after a 2-week high-sucrose diet or a 3-month high-fat diet. Even in the absence of hypertriglyceridemia, TAG increased 2-fold with both diets in hearts from wildtype mice. In contrast, hearts from Gpat1 −/− mice contained 20–80% less TAG than the wildtype controls. In addition, hearts from Gpat1 −/− mice fed the high-sucrose diet incorporate 60% less [14C]palmitate into heart TAG as compared to wildtype mice. Because GPAT1 prefers 16:0-CoA to other long-chain acyl-CoA substrates, we determined the fatty acid composition of heart phospholipids. Compared to wildtype littermate controls, hearts from Gpat1 -/- −/− mice contained a lower amount of 16:0 in phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine/phosphatidylinositol and significantly more C20:4n6. Phosphatidylcholine and phosphatidylethanolamine from Gpat1 -/- −/− hearts also contained higher amounts of 18:0 and 18:1. Although at least three other GPAT isoforms are expressed in the heart, our data suggest that GPAT1 contributes significantly to cardiomyocyte TAG synthesis during lipogenic or high-fat diets and influences the incorporation of 20:4n6 into heart phospholipids.
Keywords: Obesity; Type 2 diabetes; Lipotoxicity; Diabetic cardiomyopathy; Arachidonic acid;