BBA - Molecular and Cell Biology of Lipids (v.1801, #4)

2-Hydroxy fatty acids (hFA) are important components of a subset of mammalian sphingolipids. The presence of hFA in sphingolipids is best described in the nervous system, epidermis, and kidney. However, the literature also indicates that various hFA-sphingolipids are present in additional tissues and cell types, as well as in tumors. Biosynthesis of hFA-sphingolipids requires fatty acid 2-hydroyxlase, and degradation of hFA-sphingolipids depends, at least in part, on lysosomal acid ceramidase and the peroxisomal fatty acid α-oxidation pathway. Mutations in the fatty acid 2-hydroxylase gene, FA2H, have been associated with leukodystrophy and spastic paraparesis in humans, underscoring the importance of hFA-sphingolipids in the nervous system. In the epidermis, hFA-ceramides are essential for the permeability barrier function. Physiological function of hFA-sphingolipids in other organs remains largely unknown. Recent evidence indicates that hFA-sphingolipids have specific roles in cell signaling.
Keywords: Fatty acid 2-hydroxylase; Fatty acid alpha-hydroxylase; FA2H; Hydroxy fatty acid; Hydroxy sphingolipids;

Modulation of plasma TG lipolysis by Angiopoietin-like proteins and GPIHBP1 by Laeticia Lichtenstein; Sander Kersten (415-420).
There is evidence that elevated plasma triglycerides (TG) serve as an independent risk factor for coronary heart disease. Plasma TG levels are determined by the balance between the rate of production of chylomicrons and VLDL in intestine and liver, respectively, and their rate of clearance in peripheral tissues. Lipolytic processing of TG-rich lipoproteins is mediated by the enzyme lipoprotein lipase (LPL), which is tethered to the capillary endothelium via heparin sulphate proteoglycans. In recent years the Angiopoietin-like proteins ANGPTL3 and ANGPTL4 have emerged as novel modulators of LPL activity. Studies in transgenic animals supported by in vitro experiments have demonstrated that ANGPTL3 and ANGPTL4 impair plasma TG clearance by inhibiting LPL activity. In humans, genetic variation within the ANGPTL3 and ANGPTL4 genes contributes to variation in plasma TG and HDL levels, thereby validating the importance of ANGPTLs in the regulation of lipoprotein metabolism in humans. Combined with the discovery of GPIHBP1 as a likely LPL anchor, these findings have led to a readjustment of the mechanism of LPL function. This review provides an overview of our current understanding of the role and regulation of ANGPTL3, ANGPTL4 and GPIHBP1, and places the newly acquired knowledge in the context of the established function and mechanism of LPL-mediated lipolysis.
Keywords: Lipoprotein; Triglyceride; Angiopoietin-like protein; GPIHBP1;

ADD1/SREBP1c activates the PGC1-α promoter in brown adipocytes by Qin Hao; Jacob B. Hansen; Rasmus K. Petersen; Philip Hallenborg; Claus Jørgensen; Saverio Cinti; Philip J. Larsen; Knut R. Steffensen; Haibo Wang; Sheila Collins; Jun Wang; Jan-Åke Gustafsson; Lise Madsen; Karsten Kristiansen (421-429).
Cold adaptation elicits a paradoxical simultaneous induction of fatty acid synthesis and β-oxidation in brown adipose tissue. We show here that cold exposure coordinately induced liver X receptor α (LXRα), adipocyte determination and differentiation-dependent factor 1 (ADD1)/sterol regulatory element-binding protein-1c (SREBP1c) and peroxisome proliferator-activated receptor γ coactivator-1α (PGC1α) in brown and inguinal white adipose tissues, but not in epididymal white adipose tissue. Using in vitro models of white and brown adipocytes we demonstrate that β-adrenergic stimulation induced expression of LXRα, ADD1/SREBP1c and PGC1α in cells with a brown-like adipose phenotype. We demonstrate that ADD1/SREBP1c is a powerful inducer of PGC1α expression via a conserved E box in the proximal promoter and that β-adrenergic stimulation led to recruitment of ADD1/SREBP1c to this E box. The ability of ADD1/SREBP1c to activate the PGC1α promoter exhibited a striking cell type dependency, suggesting that additional cell type-restricted factors contribute to ADD1/SREBP1c-mediated activation. In conclusion, our data demonstrate a novel role of ADD1/SREBP1c as a regulator of PGC1α expression in brown adipose tissue.
Keywords: LXR; ADD1/SREBP1c; PGC1α; Adipogenesis; Brown adipocytes;

Electronegative LDL induction of apoptosis in macrophages: Involvement of Nrf2 by A.M.C. Pedrosa; L.A. Faine; D.M. Grosso; B. de Las Heras; L. Boscá; D.S.P. Abdalla (430-437).
The aim of this study was to determine the apoptotic pathways and mechanisms involved in electronegative LDL [LDL(−)]-induced apoptosis in RAW 264.7 macrophages and the role of Nrf2 in this process. Incubation of RAW 264.7 macrophages with LDL(−) for 24 h resulted in dose-dependent cell death. Activated caspases were shown to be involved in the apoptosis induced by LDL(−); incubation with the broad caspase inhibitor z-VAD prevented apoptosis in LDL(−)-treated cells. CD95 (Fas), CD95 ligand (FasL), CD36 and the tumor necrosis factor (TNF) ligand Tnfsf10 were overexpressed in LDL(−)-treated cells. However, Bax, Bcl-2 and Mcl-1 protein levels remained unchanged after LDL(−) treatment. LDL(−) promoted hyperpolarization of the mitochondrial membrane, elevated reactive oxygen species (ROS) production and translocation of Nrf2 to the nucleus, a process absent in cells treated with native LDL. Elicited peritoneal macrophages from Nrf2-deficient mice exhibited an elevated apoptotic response after challenge with LDL(−), together with an increase in the production of ROS in the absence of alterations in CD36 expression. These results provide evidence that CD36 expression induced by LDL(−) is Nrf2-dependent. Also, it was demonstrated that Nrf2 acts as a compensatory mechanism of LDL(−)-induced apoptosis in macrophages.
Keywords: Electronegative LDL; Nrf2; RAW macrophages; Apoptosis; Fas ligand;

A novel function of the human CLS1 in phosphatidylglycerol synthesis and remodeling by Jia Nie; Xinbao Hao; Daohong Chen; Xiao Han; Zhijie Chang; Yuguang Shi (438-445).
Phosphatidylglycerol (PG) is a precursor for the biosynthesis of cardiolipin and a signaling molecule required for various cellular functions. PG is subjected to remodeling subsequent to its de novo biosynthesis in mitochondria to incorporate appropriate acyl content for its biological functions and to prevent the harmful effect of lysophosphatidylglycerol (LPG) accumulation. Yet, a gene encoding a mitochondrial LPG acyltransferase has not been identified. In this report, we identified a novel function of the human cardiolipin synthase (hCLS1) in regulating PG remodeling. In addition to the reported cardiolipin synthase activity, the recombinant hCLS1 protein expressed in COS-7 cells and Sf-9 insect cells exhibited a strong acyl-CoA-dependent LPG acyltransferase activity, which was further confirmed by purified hCLS1 protein overexpressed in Sf-9 cells. The recombinant hCLS1 displayed an acyl selectivity profile in the order of in the order of C18:1 > C18:2 > C18:0 > C16:0, which is similar to that of hCLS1 toward PGs in cardiolipin synthesis, suggesting that the PG remodeling by hCLS1 is an intrinsic property of the enzyme. In contrast, no significant acyltransferase activity was detected from the recombinant hCLS1 enzyme toward lysocardiolipin which shares a similar structure with LPG. In support of a key function of hCLS1 in PG remodeling, overexpression of hCLS1 in COS-7 cells significantly increased PG biosynthesis concurrent with elevated levels of cardiolipin without any significant effects on the biosynthesis of other phospholipids. These results demonstrate for the first time that hCLS1 catalyzes two consecutive steps in cardiolipin biosynthesis by acylating LPG to PG and then converting PG to cardiolipin.
Keywords: Lysophosphatidylglycerol; Lysocardiolipin; Acyltransferase; Cardiolipin; Synthase;

Choline kinase in mammals is encoded by two genes, Chka and Chkb. Disruption of murine Chka leads to embryonic lethality, whereas a spontaneous genomic deletion in murine Chkb results in neonatal forelimb bone deformity and hindlimb muscular dystrophy. Surprisingly, muscular dystrophy isn't significantly developed in the forelimb. We have investigated the mechanism by which a lack of choline kinase β, encoded by Chkb, results in minimal muscular dystrophy in forelimbs. We have found that choline kinase β is the major isoform in hindlimb muscle and contributes more to choline kinase activity, while choline kinase α is predominant in forelimb muscle and contributes more to choline kinase activity. Although choline kinase activity is decreased in forelimb muscles of Chkb −/− mice, the activity of CTP:phosphocholine cytidylyltransferase is increased, resulting in enhanced phosphatidylcholine biosynthesis. The activity of phosphatidylcholine phospholipase C is up-regulated while the activity of phospholipase A2 in forelimb muscle is not altered. Regeneration of forelimb muscles of Chkb −/− mice is normal when challenged with cardiotoxin. In contrast to hindlimb muscle, mega-mitochondria are not significantly formed in forelimb muscle of Chkb −/− mice. We conclude that the relative lack of muscle degeneration in forelimbs of Chkb −/− mice is due to abundant choline kinase α and the stable homeostasis of phosphatidylcholine.
Keywords: Phosphatidylcholine; Choline kinase; Muscle; CTP:phosphocholine cytidylyltransferase; Muscular dystrophy;

Functional characterization of lysophosphatidic acid phosphatase from Arabidopsis thaliana by Venky Sreedhar Reddy; D.K. Venkata Rao; Ram Rajasekharan (455-461).
Lysophosphatidic acid (LPA) acts as a signaling molecule that regulates diverse cellular processes and it can rapidly be metabolized by phosphatase and acyltransferase. LPA phosphatase gene has not been identified and characterized in plants so far. The BLAST search revealed that the At3g03520 is similar to phospholipase family, and distantly related to bacterial phosphatases. The conserved motif, (J)4XXXNXSFD, was identified in both At3g03520 like phospholipases and acid phosphatases. In silico expression analysis of At3g03520 revealed a high expression during phosphate starvation and abiotic stresses. This gene was overexpressed in Escherichia coli and shown to posses LPA specific phosphatase activity. These results suggest that this gene possibly plays a role in signal transduction and storage lipid synthesis.
Keywords: Lysophosphatidic acid phosphatase; Lipid metabolism; Phylogenetic analysis; Soluble triacylglycerol biosynthesis; Phospholipases; Conserved motif;

Hyperforin induces Ca2+-independent arachidonic acid release in human platelets by facilitating cytosolic phospholipase A2 activation through select phospholipid interactions by Marika Hoffmann; Jakob J. Lopez; Carlo Pergola; Christian Feisst; Sven Pawelczik; Per-Johan Jakobsson; Bernd L. Sorg; Clemens Glaubitz; Dieter Steinhilber; Oliver Werz (462-472).
Here, we investigated the modulation of cytosolic phospholipase A2 (cPLA2)-mediated arachidonic acid (AA) release by the polyprenylated acylphloroglucinol hyperforin. Hyperforin increased AA release from human platelets up to 2.6 fold (maximal effect at 10 µM) versus unstimulated cells, which was blocked by cPLA2α-inhibition, and induced translocation of cPLA2 to a membrane compartment. Interestingly, these stimulatory effects of hyperforin were even more pronounced after depletion of intracellular Ca2+ by EDTA plus BAPTA/AM. Hyperforin induced phosphorylation of cPLA2 at Ser505 and activated p38 mitogen-activated protein kinase (MAPK), and inhibition of p38 MAPK by SB203580 prevented cPLA2 phosphorylation. However, neither AA release nor translocation of cPLA2 was abrogated by SB203580. In cell-free assays using liposomes prepared from different lipids, hyperforin failed to stimulate phospholipid hydrolysis by isolated cPLA2 in the presence of Ca2+. However, when Ca2+ was omitted, hyperforin caused a prominent increase in cPLA2 activity using liposomes composed of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphoethanolamine but not of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (PAPC) unless the PAPC liposomes were enriched in cholesterol (20 to 50%). Finally, two-dimensional 1H-MAS-NMR analysis visualized the directed insertion of hyperforin into POPC liposomes. Together, hyperforin, through insertion into phospholipids, may facilitate cPLA2 activation by enabling its access towards select lipid membranes independent of Ca2+ ions. Such Ca2+- and phosphorylation-independent mechanism of cPLA2 activation may apply also to other membrane-interfering molecules.
Keywords: Phospholipases A2; Arachidonic acid; Platelets; Hyperforin; Calcium; Phosphorylation;

α1-Fetoprotein transcription factor (FTF)/liver receptor homolog-1 (LRH-1) is an essential lipogenic regulator by Zhumei Xu; Lingli Ouyang; Antonio del Castillo-Olivares; William M. Pandak; Gregorio Gil (473-479).
α1-Fetoprotein transcription factor (FTF), also known as liver receptor homolog 1 (LRH-1) is highly expressed in the liver and intestine, where it is implicated in the regulation of cholesterol, bile acid and steroid hormone homeostasis. FTF is an important regulator of bile acid metabolism. We show here that FTF plays a key regulatory role in lipid homeostasis including triglyceride and cholesterol homeostasis. FTF deficient mice developed lower levels of serum triglyceride and cholesterol as a result of lower expression of several hepatic FTF target genes. Chenodeoxycholic acid repressed FTF expression resulting in a decrease in serum triglyceride in wild-type mice. The absence of chenodeoxycholic acid-mediated repression in FTF+/ mice demonstrated the essential role of FTF in triglyceride metabolism. Taken together, our results identify the nuclear receptor FTF as a central regulator of lipid metabolism.
Keywords: Triglyceride; Cholesterol; Bile acid; Nuclear receptor; Knockout mice;

Phosphatidylethanolamine synthesized by four different pathways is supplied to the plasma membrane of the yeast Saccharomyces cerevisiae by Irmgard Schuiki; Martina Schnabl; Tibor Czabany; Claudia Hrastnik; Günther Daum (480-486).
In this study, we examined the contribution of the four different pathways of phosphatidylethanolamine (PE) synthesis in the yeast Saccharomyces cerevisiae to the supply of this phospholipid to the plasma membrane. These pathways of PE formation are decarboxylation of phosphatidylserine (PS) by (i) phosphatidylserine decarboxylase 1 (Psd1p) in mitochondria and (ii) phosphatidylserine decarboxylase 2 (Psd2p) in a Golgi/vacuolar compartment, (iii) incorporation of exogenous ethanolamine and ethanolamine phosphate derived from sphingolipid catabolism via the CDP-ethanolamine pathway in the endoplasmic reticulum (ER), and (iv) synthesis of PE through acylation of lyso-PE catalyzed by the acyl-CoA-dependent acyltransferase Ale1p in the mitochondria associated endoplasmic reticulum membrane (MAM). Deletion of PSD1 and/or PSD2 led to depletion of total cellular and plasma membrane PE level, whereas mutation in the other pathways had practically no effect. Analysis of wild type and mutants, however, revealed that all four routes of PE synthesis contributed not only to PE formation but also to the supply of PE to the plasma membrane. Pulse-chase labeling experiments with L[3H(G)]serine and [14C]ethanolamine confirmed the latter finding. Fatty acid profiling demonstrated a rather balanced incorporation of PE species into the plasma membrane irrespective of mutations suggesting that all four pathways of PE synthesis provide at least a basic portion of “correct” PE species required for plasma membrane biogenesis. In summary, the PE level in the plasma membrane is strongly influenced by total cellular PE synthesis, but fine tuned by selective assembly mechanisms.
Keywords: Phosphatidylethanolamine; Phospholipid; Fatty acid; Plasma membrane; Yeast;

Low-density lipoprotein and oxysterols suppress the transcription of CTP:Phosphoethanolamine cytidylyltransferase in vitro by Hiromi Ando; Yasuhiro Horibata; Satoko Yamashita; Tetsunari Oyama; Hiroyuki Sugimoto (487-495).
The rate-limiting step in phosphatidylethanolamine (PE) synthesis by the CDP-ethanolamine pathway is the second step, which is catalyzed by CTP:phosphoethanolamine cytidylyltransferase (ET). The rate-limiting step for phosphatidylcholine biosynthesis by the CDP-choline pathway is also the second step, which is catalyzed by CTP:phosphocholine cytidylyltransferase (CT). The transcription of the most active form of CT, CTα, in serum-starved cells was stimulated by fetal bovine serum (FBS). Therefore, we were interested in the effects of FBS on the transcription of ET. Unexpectedly, the ET mRNA levels were significantly increased after NIH3T3 cells were cultured in serum-starved medium (0.5% FBS) longer than 8 h, and the increase was suppressed by the addition of FBS. Actinomycin-D inhibited the increased ET mRNA levels in serum-starved cells. ET enzyme activities and protein amounts were also increased after serum starvation. These results suggest that FBS contains substances that inhibit the transcription of ET. To identify these substances, cells were incubated with several fractions of FBS separated by molecular sizes. As expected from the results, low-density lipoprotein, 25-hydroxycholesterol (25-OHC), 24-OHC, 27-OHC, 24(S),25-epoxycholesterol and mevalonolactate suppressed the ET mRNA levels in serum-starved cells, similar to 3-hydroxy-3-methylglutaryl-CoA reductase but not CTα. These results suggest that oxysterols are important regulating lipids for the suppression of ET transcription and may help maintain the contents of PE and cholesterol at the same ratio in the cellular membrane.
Keywords: CTP:phosphoethanolamine cytidylyltransferase; Oxysterol; 25-hydroxycholesterol; Phosphatidylethanolamine; Fetal bovine serum; Low-density lipoprotein; HMG-CoA reductase; CTP:phosphocholine cytidylyltransferase;

Phosphatidylcholine transfer protein (PC-TP, a.k.a. StarD2) is abundantly expressed in liver and is regulated by PPARα. When fed the synthetic PPARα ligand fenofibrate, Pctp −/− mice exhibited altered lipid and glucose metabolism. Microarray profiling of livers from fenofibrate fed wild type and Pctp −/− mice revealed differential expression of a broad array of metabolic genes, as well as their regulatory transcription factors. PC-TP expression in cell culture controlled the activities of both PPARα and HNF4α, suggesting that the mechanism by which it modulates hepatic metabolism is at least in part via activation of transcription factors that govern nutrient homeostasis.
Keywords: START domain; Fibrate drug; Lipid; Glucose; Microarray; Gene transcription;

Reaction mechanism of 5,8-linoleate diol synthase, 10R-dioxygenase, and 8,11-hydroperoxide isomerase of Aspergillus clavatus by Fredrik Jernerén; Ulrike Garscha; Inga Hoffmann; Mats Hamberg; Ernst H. Oliw (503-507).
Aspergilli express fusion proteins of an animal haem peroxidase domain with fatty acid dioxygenase (DOX) activity (∼ 600 amino acids) and a functional or non-functional hydroperoxide isomerase/cytochrome P450 domain (∼ 500 amino acids with EXXR and GPHXCLG motifs). 5,8-Linoleate diol synthases (LDS; ppoA) and 10R-DOX (ppoC) of A spergillus nidulans and A. fumigatus belong to this group. Our objective was to determine the oxylipins formed from linoleic acid by A. clavatus and their mechanism of biosynthesis. A. clavatus oxidized linoleic acid to (8R)-hydroperoxylinoleic acid (8R-HPODE), (10R)-hydroperoxy-8(E),12(Z)-octadecadienoic acid (10R-HPODE), and to (5S,8R)-dihydroxy- and (8R,11S)-dihydroxylinoleic acids (DiHODE) as major products. This occurred by abstraction of the pro-S hydrogen at C-8 and antarafacial dioxygenation at C-8 or at C-10 with double bond migration. 8R-HPODE was then isomerized to 5S,8R-DiHODE and to 8R,11S-DiHODE by abstraction of the pro-S hydrogens at C-5 and C-11 of 8R-HPODE, respectively, followed by suprafacial oxygenation. The genome of A. clavatus codes for two enzymes, which can be aligned with > 65% amino acid identity to 10R-DOX and 5,8-LDS, respectively. The 5,8-LDS homologue likely forms and isomerizes 8R-HPODE to 5S,8R-DiHODE. A third gene (ppoB) codes for a protein which carries a serine residue at the cysteine position of the P450 motif. This Cys to Ser replacement is known to abolish P450 2B4 catalysis and the hydroperoxide isomerase activity of 5,8-LDS, suggesting that ppoB of A. clavatus may not be involved in the biosynthesis of 8R,11S-DiHODE.
Keywords: Dioxygenase; Hydroperoxide isomerase; Myeloperoxidase; Oxygenation mechanism; Cytochrome P450;

Lipolysis of natural long chain and synthetic medium chain galactolipids by pancreatic lipase-related protein 2 by Sawsan Amara; Nathalie Barouh; Jérôme Lecomte; Dominique Lafont; Sylvie Robert; Pierre Villeneuve; Alain De Caro; Frédéric Carrière (508-516).
Monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the most abundant lipids in nature, mainly as important components of plant leaves and chloroplast membranes. Pancreatic lipase-related protein 2 (PLRP2) was previously found to express galactolipase activity, and it is assumed to be the main enzyme involved in the digestion of these common vegetable lipids in the gastrointestinal tract. Most of the previous in vitro studies were however performed with medium chain synthetic galactolipids as substrates. It was shown here that recombinant guinea pig (Cavia porcellus) as well as human PLRP2 hydrolyzed at high rates natural DGDG and MGDG extracted from spinach leaves. Their specific activities were estimated by combining the pH-stat technique, thin layer chromatography coupled to scanning densitometry and gas chromatography. The optimum assay conditions for hydrolysis of these natural long chain galactolipids were investigated and the optimum bile salt to substrate ratio was found to be different from that established with synthetic medium chains MGDG and DGDG. Nevertheless the length of acyl chains and the nature of the galactosyl polar head of the galactolipid did not have major effects on the specific activities of PLRP2, which were found to be very high on both medium chain [1786 ± 100 to 5420 ± 85 U/mg] and long chain [1756 ± 208 to 4167 ± 167 U/mg] galactolipids. Fatty acid composition analysis of natural MGDG, DGDG and their lipolysis products revealed that PLRP2 only hydrolyzed one ester bond at the sn - 1 position of galactolipids. PLRP2 might be used to produce lipid and free fatty acid fractions enriched in either 16:3 n  − 3 or 18:3 n  − 3 fatty acids, both found at high levels in galactolipids.
Keywords: Bile salt; Digalactosyl diacylglycerol; Fatty acid; Galactolipase; Galactolipids; Gas chromatography; Mixed micelle; Monogalactosyl diacylglycerol; Pancreatic lipase-related protein 2; Thin layer chromatography;

Intestinal lipid alterations occur prior to antibody-induced prostaglandin E2 production in a mouse model of ischemia/reperfusion by Byron L. Sparkes; Emily E. Archer Slone; Mary Roth; Ruth Welti; Sherry D. Fleming (517-525).
Ischemia/reperfusion (IR) induced injury results in significant tissue damage in wild-type, but not antibody-deficient, Rag-1−/− mice. However, Rag-1−/− mice sustain intestinal damage after administration of wild-type antibodies or naturally occurring, specific anti-phospholipid related monoclonal antibodies, suggesting involvement of a lipid antigen. We hypothesized that IR initiates metabolism of cellular lipids, resulting in production of an antigen recognized by anti-phospholipid antibodies. At multiple time points after Sham or IR treatment, lipids extracted from mouse jejunal sections were analyzed by electrospray ionization triple quadrupole mass spectrometry. Within 15 min of reperfusion, IR induced significantly more lysophosphatidylcholine (lysoPC), lysophosphatidylglycerol (lysoPG) and free arachidonic acid (AA) production than Sham treatment. While lysoPC, lysoPG, and free AA levels were similar in C57Bl/6 (wild-type) and Rag-1−/− mice, IR led to Cox-2 activation and prostaglandin E2 (PGE2) production in wild-type, but not in the antibody-deficient, Rag-1−/− mice. Administration of wild-type antibodies to Rag-1−/− mice restored PGE2 production and intestinal damage. These data indicate that IR-induced intestinal damage requires antibodies for Cox-2 stimulated PGE2 production but not for production of lysoPC and free AA.
Keywords: Mass spectrometry; Mice; Intestine; Lipidomics;

The ability of n-3 PUFA to competitively inhibit the use of arachidonic acid (AA) for membrane phospholipid synthesis and prostaglandin E2 (PGE2) production has been well demonstrated in single cell models. In the present study, we investigated the metabolic competition between AA and eicosapentaenoic acid (EPA) for PGE2 synthesis in a rat hepatocyte–Kupffer cell (HPC/KC) co-culture system when the cellular oxidation capacity was enhanced by exogenous l-carnitine. We demonstrate that in the absence of l-carnitine, 1) β-oxidation rates of EPA and AA were comparable in HPCs and in KCs; 2) AA and not EPA was preferentially incorporated into glycerolipids; and 3) addition of EPA significantly decreased AA-dependent PGE2 synthesis in HPCs and cyclooxygenase-2 (COX-2) expression in co-cultured HPCs/KCs. However, enhancing the cellular oxidation capacity by the addition of l-carnitine 1) significantly increased β-oxidation of EPA in HPCs, but only marginally elevated the oxidation of AA in HPCs and the oxidation of both fatty acids in KCs; 2) decreased the esterification, but did not alter the preferential incorporation of AA into glycerolipids; and 3) alleviated the significant competitive inhibition of AA-dependent PGE2 synthesis and COX-2 expression by EPA. Taken together, the results strongly suggest that l-carnitine affects competition between AA and EPA in PG synthesis in liver cells by enhancing oxidation of EPA in HPCs. This implies that the beneficial effects of n-3 PUFA, especially EPA, are affected by the cellular oxidation capacity.
Keywords: l-Carnitine; Eicosapentaenoic acid; Arachidonic acid; Prostaglandin E2; β-oxidation; Hepatocyte–Kupffer cell co-culture;

CTP:phosphocholine cytidylyltransferase alpha (CCTα) is a key enzyme for phosphatidylcholine biosynthesis in mammalian cells. This enzyme plays an essential role in all processes that require membrane biosynthesis such as cell proliferation and viability. Thus, CCTα activity and expression fluctuate during the cell cycle to achieve PtdCho requirements. We demonstrated, for the first time, that CCTα is localized in the nucleus in cells transiting the S phase, whereas it is localized in the cytoplasm of G0-arrested cells, suggesting a specific role of nuclear CCTα during the S phase. We also investigated how E2F1 influences the regulation of the CCTα-promoter during the S phase; we demonstrated that E2F1 is necessary, but not sufficient, to activate CCTα expression when this factor is over-expressed. However, when E2F1 and Sp1 were over-expressed, the transcription from the CCTα-promoter reporter construct was super-activated. Transient transfection studies demonstrated that E2F1 could super-activate Sp1-dependent transcription in a promoter containing only the Sp1 binding sites “B” or “C”, and that Sp1 could activate Sp1-dependent transcription in a promoter containing the E2F site, thus, further demonstrating a functional interaction of these factors. In conclusion, the present results allowed us to portray the clearest picture of the CCTα-gene expression in proliferating cells, and understand the mechanism by which cells coordinate cell cycle progression with the requirement for phosphatidylcholine.
Keywords: Pcyt1a gene promoter; Cytidylyltransferase; Phosphatidylcholine; E2F1; Sp1; S phase;