BBA - Molecular and Cell Biology of Lipids (v.1683, #1-3)

Tyrosine nitration of carnitine palmitoyl transferase I during endotoxaemia in suckling rats by Koji Fukumoto; Agostino Pierro; Victor A Zammit; Lewis Spitz; Simon Eaton (1-6).
Heart carnitine palmitoyl transferase I (CPTI) is inhibited in vivo during endotoxaemia and in vitro by peroxynitrite but the biochemical basis of this inhibition is not known. The aim of this study was to determine which isoform of CPT I is inhibited during endotoxaemia and whether the inhibition is due to increased tyrosine nitration. Cardiac mitochondria were isolated from endotoxaemic suckling rats. To determine whether M- or L-CPTI was inhibited, we carried out titrations with DNP-etomoxir-CoA. Slopes of the titration curves with DNP-etomoxir-CoA were no different between control and endotoxaemia, suggesting that M-CPTI was specifically inhibited. Immunoprecipitation was carried out using an anti-nitrotyrosine antibody. Immunoprecipitated proteins were identified by Western blotting with L- and M-CPTI specific antibodies. L-CPTI was nitrated both in control and in 2- and 6-h endotoxaemia mitochondria but there was no significant difference in the level of nitration. M-CPTI was also nitrated in control mitochondria but nitration was significantly increased at both 2- and 6-h endotoxaemia. Either 10 mM 3-nitrotyrosine plus 40 μg nitrated-albumin or 0.5 M dithionite, during immunoprecipitation, greatly decreased immunopositivity for M- and L-CPTI on WB. M-CPTI appears to be a novel target for peroxynitrite during endotoxaemia, which would alter myocardial substrate selection.
Keywords: Sepsis; Fatty acid oxidation; Carnitine palmitoyl transferase I; Nitration; Peroxynitrite;

Expression and induction of CYP4F subfamily in human leukocytes and HL60 cells by Yasushi Kikuta; Yoshiaki Yamashita; Soichiro Kashiwagi; Kazunori Tani; Kazushi Okada; Kiyofumi Nakata (7-15).
We investigated the expression of the CYP4F subfamily in human leukocytes and HL60 cells. Enzymatic activity assay, immunocytochemical staining, and reverse transcription-polymerase chain reaction (RT-PCR) analysis of human leukocytes showed that polymorphonuclear leukocytes (PMNs) expressed CYP4F3B and CYP4F12 in addition to CYP4F3. Transcription start site of CYP4F3B mRNA in the leukocytes was identical to that of CYP4F3 mRNA. The HL60 cells, which were differentiated into PMN-like shapes by treatment with all-trans-retinoic acid (RA), also expressed CYP4F3, CYP4F3B and CYP4F12. CYP4F3 was expressed in one third of the peripheral monocytes, which ω-hydroxylated leukotriene B4 (LTB4) at a rate 11 times lower than that of PMN. The cells that were differentiated into a form similar to monocytes/macrophages in shape by treatment with 12-myristate 13-acetate expressed mRNA for CYP4F3 and CYP4F3B. Promoter analysis of the CYP4F3 gene demonstrated that a region (−174/−90) of this gene was important for its promoter activity in the HL60 cells. This is the first report on the distribution of different CYP4F isoforms in leukocytes and their induction in HL60 cells.
Keywords: Cytochrome P450; Leukocyte; HL60 cell; Leukotriene B4; ω-Hydroxylase;

The extraordinary antioxidant activity of vitamin E phosphate by Bashir M Rezk; Guido R.M.M Haenen; Wim J.F van der Vijgh; Aalt Bast (16-21).
The antioxidant activities of RRR-vitamin E (VE), all-rac-vitamin E (all-rac-VE), trolox, RRR-vitamin E acetate (VEA), all-rac-vitamin E phosphate (VEP) and RRR-vitamin E succinate (VES) were compared. In this study, the rank order in the inhibition of lipid peroxidation (LPO) of VE and its derivatives was trolox>VE≈all-rac-VE>VEA>VES. VE and trolox inhibited LPO in non-heated and heated rat liver microsomes. It has generally been accepted that this is due to scavenging of free radicals by these antioxidants, and during this protection the antioxidants are oxidized. VEA and VES have to be converted into VE by esterases to obtain antioxidant activity against LPO. VEP, however, had a potent antioxidant effect of its own without conversion to VE. In contrast to VE, VEP is not consumed during this protection. Of the compounds tested, VEP is the most potent in induction of hemolysis of erythrocytes. EPR experiments using the spin label 16-doxylstearic acid showed that VEP reduces membrane fluidity, in contrast to VE. This indicates that VEP acts as a detergent and forms a barrier that might inhibit the transfer of radicals from one polyunsaturated fatty acid to another. This new mechanism may form the basis for a new class of antioxidants.
Keywords: Vitamin E; Trolox; Vitamin E acetate; Vitamin E phosphate; Vitamin E succinate; Antioxidant activity; Microsome;

Induction of antioxidant enzymes by FAK in a human leukemic cell line, HL-60 by Yoshiko Sonoda; Naomi Aiba; Ryouko Utsubo; Emiko Koguchi; Maki Hasegawa; Tadashi Kasahara (22-32).
We have established several focal adhesion kinase (FAK) cDNA-transfected HL-60 (HL-60/FAK) cells which were highly resistant to oxidative stress-induced apoptosis. To identify target genes that are involved in HL-60/FAK cells, we performed cDNA microarray screening using apoptosis-chip. There, we identified the decrease of glutathione peroxidase (GPx). This result prompted us to investigate the changes of antioxidant enzymes. Here, we demonstrate that lipid peroxidation was suppressed after treatment with hydrogen peroxide in HL-60/FAK cells but not vector-transfected HL-60 (HL-60/Vect) cells. Furthermore, we demonstrate that HL-60/FAK cells have higher basal reactive oxygen species (ROS) levels than the parental HL-60 or HL-60/Vect cells, while ROS accumulation by hydrogen peroxide treatment was almost the same in these cells. Basal activity and mRNA expression of antioxidant enzymes, particularly of GSH reductase (GRe), phospholipid hydroperoxide glutathione peroxidase (PHGPx) were markedly elevated in HL-60/FAK cells. In contrast, GPx and catalase levels were decreased in HL-60/FAK cells. Further, a Src family kinases inhibitor, PP2, suppressed GRe and PHGPx mRNA by inactivation of FAK and c-Src in HL-60/FAK cells. These results suggest that FAK upregulates antioxidant enzymes and suppresses lipid peroxidation, resulting in the anti-apoptotic state for oxidative stress.
Keywords: FAK; Anti-apoptosis; ROS; Antioxidant enzyme; Lipid peroxidation; PHGPx;

Transforming growth factor-beta down-regulates apolipoprotein M in HepG2 cells by Ning Xu; Maria Hurtig; Xiao-Ying Zhang; Qing Ye; Peter Nilsson-Ehle (33-37).
Apolipoprotein M (apoM) is a novel apolipoprotein presented mostly in high-density lipoprotein (HDL) in human plasma, and is exclusively expressed in liver and in kidney. The pathophysiological function of apoM has not yet been elucidated. Apolipoprotein B (apoB), the characteristic apolipoprotein of low-density lipoprotein (LDL), is like apoM, a very hydrophobic protein, and thereafter they both must co-circulate with lipoprotein particles in plasma. The cytokine, transforming growth factor-beta (TGF-β), has been shown to decreased apoB secretion in HepG2 cells, and we hypothesized that TGF-β may have the same effects on apoM expression in HepG2 cells. In the present study, we used real-time RT-PCR to analyze apoM and apoB mRNA levels during administration of TGF-β, as well as TGF-α, epidermal growth factor (EGF) and hepatic growth factor (HGF). TGF-β significantly inhibited both apoM and apoB mRNA expression in HepG2 cells. The inhibitory effects of TGF-β were dose-dependent, i.e. 1 ng/ml of TGF-β decreased apoM mRNA levels by 30%, and 10 or 100 ng/ml of TGF-β decreased apoM mRNA levels more than 65%. The effect of TGF-β on apoB mRNA expression was slightly weaker than that of apoM, with a maximum effect at 10 or 100 ng/ml TGF-β where apoB mRNA levels decreased about 55%. The inhibitory effects of TGF-β on apoM and apoB mRNA levels also increased with increasing incubation time, where the maximum effect was obtained at 24 h. Moreover TGF-α, EGF and HGF all decreased both apoM and apoB mRNA levels, but to a less extent than TGF-β. Further, all four cytokines had more pronounced effects on apoM mRNA expression than apoB mRNA expression. The present study suggested that apoM, like apoB, may be involved in the hepatic lipoprotein assembly in vivo.
Keywords: Lipoprotein; Apolipoprotein B; Apolipoprotein M; TGF; EGF; HGF; HepG2 cell line;

Cyclooxygenase-2 (COX-2) is an inducible enzyme responsible for high-level prostaglandin production during inflammation and carcinogenesis. In this study, the transcriptional regulation of COX-2 expression induced by epidermal growth factor (EGF) in human epidermoid carcinoma A431 cells was studied. EGF treatment induced the expression of COX-2 mRNA, protein, promoter and enzyme activity in a time-dependent manner. EGF-induced COX-2 promoter activity was inhibited by overexpression of the dominant-negative forms of Ras and ERK2. Induction of COX-2 and c-Jun by EGF was completely suppressed by MEK inhibitor combined with JNK inhibitor. Analysis of the COX-2 promoter binding proteins by gel mobility shift assay and DNA affinity precipitation assay revealed that c-Jun and p300 binding to CRE/E-box site were responsible for the EGF-induced COX-2 gene transcription. Overexpression of p300 significantly enhanced COX-2 promoter activity in cells overexpressed of c-Jun or treated with EGF. EGF- and c-Jun-induced transcription of COX-2 promoter was repressed by cotransfection of E1A in a dose-dependent manner. All together, these results indicated that the EGF-induced expression of COX-2 in A431 cells was mediated through the Ras-ERK/JNK signaling pathway, and subsequent induction of c-Jun following MAPK activation, in cooperation with coactivator p300, was required for the EGF response.
Keywords: Cyclooxygenase-2; Mitogen-activated protein kinase; c-Jun; p300;

Effects of peroxisome proliferator-activated receptor α activation on pathways contributing to cholesterol homeostasis in rat hepatocytes by Catherine Le Jossic-Corcos; Sandrine Duclos; Leyla C Ramirez; Isabelle Zaghini; Grégory Chevillard; Pascal Martin; Thierry Pineau; Paulette Bournot (49-58).
Peroxisome proliferator-activated receptor alpha (PPARα) activation by fibrates controls expression of several genes involved in hepatic cholesterol metabolism. Other genes could be indirectly controlled in response to changes in cellular cholesterol availability. To further understand how fibrates may affect cholesterol synthesis, we investigated in parallel the changes in the metabolic pathways contributing to cholesterol homeostasis in liver. Ciprofibrate increased HMG-CoA reductase and FPP synthase mRNA levels in rat hepatocytes, together with cholesterogenesis from [14C] acetate and [3H] mevalonate. The up-regulation observed in fenofibrate- and WY-14,643-treated mice was abolished in PPARα-null mice, showing an essential role of PPARα. Among the three sterol regulatory element-binding protein (SREBP) mRNA species, only SREBP-1c level was significantly increased. In ciprofibrate-treated hepatocytes, cholesterol efflux was decreased, in parallel with cholesteryl ester storage and bile acids synthesis. As expected, AOX expression was strongly induced, supporting evidence of the peroxisome proliferation. Taken together, these results show that fibrates can cause cholesterol depletion in hepatocytes, possibly in part as a consequence of an important requirement of cholesterol for peroxisome proliferation, and increase cholesterogenesis by a compensatory phenomenon afterwards. Such cholesterogenesis regulation could occur in vivo, in species responsive to the peroxisome proliferative effect of PPARα ligands.
Keywords: HMG-CoA reductase; Cholesterol metabolism; Fibrate; Rat hepatocyte; PPARα-null mouse;

Lysophosphatidic acid induces exocytic trafficking of Na+/H+ exchanger 3 by E3KARP-dependent activation of phospholipase C by Jung Woong Choi; Whaseon Lee-Kwon; Eun Su Jeon; Yong Jung Kang; Kazuya Kawano; Hyeon Soo Kim; Pann-Ghill Suh; Mark Donowitz; Jae Ho Kim (59-68).
Lysophosphatidic acid (LPA) stimulates Na+/H+ exchanger 3 (NHE3) activity in opossum kidney proximal tubule (OK) cells by increasing the apical membrane amount of NHE3. This occurs by stimulation of exocytic trafficking of NHE3 to the apical plasma membrane by an E3KARP-dependent mechanism. However, it is still unclear how E3KARP leads to the LPA-induced exocytosis of NHE3. In the current study, we demonstrate that stable expression of exogenous E3KARP increases LPA-induced phospholipase C (PLC) activation and subsequent elevation of intracellular Ca2+ in opossum kidney proximal tubule (OK) cells. Pretreatment with U73122, a PLC inhibitor, prevented the LPA-induced NHE3 activation and the exocytic trafficking of NHE3. To understand how the elevation of intracellular Ca2+ leads to the stimulation of NHE3, we pretreated OK cells with BAPTA-AM, an intracellular Ca2+ chelator. BAPTA-AM completely blocked the LPA-induced increase of NHE3 activity and surface NHE3 amount by decreasing the LPA-induced exocytic trafficking of NHE3. Pretreatment with GF109203X, a PKC inhibitor, did not affect the percent of LPA-induced NHE3 activation and increase of surface NHE3 amount. From these results, we suggest that E3KARP plays a necessary role in LPA-induced PLC activation, and that PLC-dependent elevation of intracellular Ca2+ but not PKC activation is necessary for the LPA-induced increase of NHE3 exocytosis.
Keywords: NHE3; E3KARP; PDZ; Phospholipase C; Calcium; Exocytosis;

Human free apolipoprotein A-I and artificial pre-beta-high-density lipoprotein inhibit eNOS activity and NO release by Olivier Briand; Françoise Martin Nizard; Monique David-Dufilho; Isabelle Six; Sophie Lestavel; Annie Brunet; Jean-Charles Fruchart; Gérard Torpier; Régis Bordet; Véronique Clavey; Patrick Duriez (69-77).
Little is known about the effects of human free apolipoprotein A-I (Free-Apo A-I) and pre-beta-high density lipoprotein (pre-beta-HDL) on the endothelium function. In this study, we have investigated the effects of Free-Apo A-I and artificial pre-beta-HDL on endothelial NO synthase (eNOS) activity and on NO production by endothelial cells. Free-Apo A-I drastically inhibited NO production in human umbilical cord vein endothelial cells (HUVECs) and eNOS activity in bovine aortic endothelial cells (BAECs). Pre-beta-HDL and serum from human apolipoprotein A-I transgenic rabbits inhibited eNOS activity in BAECs but HDL3 did not. Free-Apo A-I displaced eNOS from BAEC plasma membrane towards intracellular pools without affecting eNOS activity and eNOS mass in BAEC crude homogenates. Free-Apo A-I and HDL3 did not decrease either caveolin bound to BAEC plasma membrane or caveola cholesterol content. As previously described, we showed that HDL3 directly induced endothelium-dependent relaxation of rings from rat aorta. We observed that pre-beta-HDL significantly decreased endothelium-dependent relaxation of rat aortic rings ex vivo.
Keywords: Endothelium; Nitric oxide synthase; Nitric oxide; Apolipoprotein A-I; Pre-beta-HDL; HDL;

CTP:phosphocholine cytidylyltransferase (CCT) is an enzyme critical for cellular phosphatidylcholine (PC) synthesis, converting phosphocholine and cytidine 5′-triphosphate (CTP) to CDP-choline. We have isolated a cDNA encoding an isoform of CCT from Drosophila melanogaster and expressed the recombinant native and 6×-His-tagged forms using a baculovirus expression system in Spodoptera frugiperda (Sf9) insect cells. Immunoblot using anti-phospho amino acid antibodies reveals the enzyme is phosphorylated on serine and threonine residues, but not tyrosine. The purified native enzyme exhibits a V max value of 1352±159 nmol CDP-choline/min/mg, a K m value of 0.50±0.09 mM for phosphocholine, and a K′ (Hill constant) value of 0.72±0.10 mM for CTP. The 6×-His-tagged enzyme has similar properties with a V max value of 2254±253 nmol CDP-choline/min/mg, a K m value of 0.63±0.13 mM for phosphocholine and a K′ for CTP equal to 0.81±0.20 mM. Each form of the enzyme was activated to a similar extent by synthetic PC vesicles containing 50 mol% oleate. The efficiency of lipid activation was greatest using PC vesicles containing diphosphatidylglycerol (DPG), significantly less efficient activation was seen when phosphatidylserine (PS) and phosphatidylinositol (PI) were incorporated into vesicles, and PC alone or PC vesicles containing phosphatidylethanolamine were the least efficient enzyme activators.
Keywords: Phosphatidylcholine; Cytidylyltransferase; Lipid activation; Drosophila melanogaster; Spodoptera frugiperda;

We present evidence that the ATP binding-cassette transporter-2 (ABCA2) is a sterol-responsive gene that has a role in the trafficking of low-density lipoprotein-derived free cholesterol (LDL-FC). In HepG2 cells ABCA2 was coordinately expressed with other sterol-responsive genes. Stable constitutive expression of ABCA2 in Chinese hamster ovary cells (CHOA2) was accompanied by an increase the expression of the low-density lipoprotein receptor (LDLR) and other genes involved in the regulation of cholesterol homeostasis. LDLR mRNA was elevated greater than ninefold and 3-hydroxy-3-methylglutaryl CoA synthase (HMGCoA S) expression was elevated sevenfold in CHOA2 cells. The increase in LDLR expression was regulated at the level of transcription; however, culture of CHO and CHOA2 cells in medium containing lipoprotein-deficient serum (LPDS) results in similar levels of LDLR promoter expression. No differences were measured in the dose-dependent uptake of fluorescently labeled 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchorate-LDL (DiI-LDL) between CHO and CHOA2 cells cultured in medium containing LPDS. Ultraviolet microscopy revealed a similar distribution of the DiI-LDL label in cytoplasmic vesicles. We measured an LDL dose-dependent reduction in esterification of LDL-FC in intact CHOA2 cells cultured in medium containing LPDS, however, no significant difference was measured in acylcoenzyme A:cholesterol acyltransferase (ACAT) activity in cell-free extracts of CHO and CHOA2 cells. CHO cells or CHOA2 cells treated with the hydrophobic amine, U18666A, showed similar filipin staining of unesterified cholesterol in cytoplasmic vesicles. Addition of progesterone or U18666A to CHO cells elevated ABCA2 expression. Finally, we found that ABCA2 expression was elevated in Niemann–Pick type C1 (NPC1) fibroblasts and in Familial Hypercholesterolemia (FHC) fibroblasts.
Keywords: ABC transporter; Low-density lipoprotein receptor; Chinese hamster ovary; U18666A; Progesterone; SREBP2; ACAT; SCAP;

The present study examines whether and to what extent the profiles of adipose-derived factors are altered in epididymal and subcutaneous adipose tissues of long-term fasted/refed and of fasted rats treated by recombinant leptin. Fasting was characterized by three successive metabolic phases. Minor differences in the time-course and magnitude of response were detected between the two adipose sites. Leptin, adiponectin, resistin, adiponutrin, and insulin-like growth factor-1 (IGF-1) gene expressions differentially decreased according to the fasting duration. mRNA levels reached a minimum in late fasting for these secreted factors, being decreased by 60–90% for adiponectin, resistin, and IGF-1, 95–98% for leptin and by 100% for adiponutrin. Refeeding partially or totally restored their mRNA expression in epididymal adipose. Expression levels of apolipoprotein E (ApoE), angiotensinogen (AGT), adipsin and macrophage migration inhibitory factor (MIF) were either unchanged or slightly affected. In leptin-treated rats, leptin mRNA concentrations were significantly decreased in phase 2 of fasting (by 85%) from levels in control phosphate-buffered saline (PBS)-treated rats in both tissues. Leptin treatment also decreased resistin mRNA levels (by 78% in P2L and 63% in P3L relative to control groups) in subcutaneous adipose. These data suggest that adiponectin, resistin, adiponutrin, and IGF-1 could be involved in overall energy homeostasis during prolonged fasting, as leptin is. The mechanisms that underlie the expressions of these adipose-secreted factors remain to be determined.
Keywords: Leptin; Adiponectin; Resistin; Adiponutrin; mRNA level; Prolonged food deprivation;