BBA - Molecular and Cell Biology of Lipids (v.1687, #1-3)
Editorial Board (ii).
The apolipoprotein(a) component of lipoprotein(a) mediates binding to laminin: contribution to selective retention of lipoprotein(a) in atherosclerotic lesions by Angela D'Angelo; Diego Geroldi; Mark A. Hancock; Viviana Valtulina; Antonia I. Cornaglia; Craig A. Spencer; Enzo Emanuele; Alberto Calligaro; Marlys L. Koschinsky; Pietro Speziale; Livia Visai (1-10).
Lipoprotein(a) [Lp(a)] entrapment by vascular extracellular matrix may be important in atherogenesis. We sought to determine whether laminin, a major component of the basal membrane, may contribute to Lp(a) retention in the arterial wall. First, immunohistochemistry experiments were performed to examine the relative distribution of Lp(a) and laminin in human carotid artery specimens. There was a high degree of co-localization of Lp(a) and laminin in atherosclerotic specimens, but not in non-atherosclerotic sections. We then studied the binding interaction between Lp(a) and laminin in vitro. ELISA experiments showed that native Lp(a) particles and 17K and 12K recombinant apolipoprotein(a) [r-apo(a)] variants interacted strongly with laminin whereas LDL, apoB-100, and the truncated KIV6-P, KIV8-P, and KIV9-P r-apo(a) variants did not. Overall, the ELISA data demonstrated that Lp(a) binding to laminin is mediated by apo(a) and a combination of the lysine analogue ε-aminocaproic acid and salt effectively decreases apo(a) binding to laminin. Secondary binding analyses with 125I-labeled r-apo(a) revealed equilibrium dissociation constants (K d) of 180 and 360 nM for the 17K and 12K variants binding to laminin, respectively. Such similar K d values between these two r-apo(a) variants suggest that isoform size does not appear to influence apo(a) binding to laminin. In summary, our data suggest that laminin may bind to apo(a) in the atherosclerotic intima, thus contributing to the selective retention of Lp(a) in this milieu.
Keywords: Lipoprotein(a); Apolipoprotein(a); Laminin; Atherosclerosis;
Impaired lipid accumulation by trans10, cis12 CLA during adipocyte differentiation is dependent on timing and length of treatment by Linda Granlund; Jan I. Pedersen; Hilde I. Nebb (11-22).
Conjugated linoleic acids (CLAs) are a group of polyunsaturated fatty acids found in ruminant products, where the predominant isomers are cis9, trans11 (c9,t11) and trans10, cis12 (t10,c12) CLA. We have previously shown that t10,c12 CLA prevents lipid accumulation in mature adipocytes in part by acting as a peroxisome proliferator-activated receptor γ (PPARγ) modulator. The objective of this study was to further establish the molecular mechanisms underlying the attenuating effect on lipid accumulation by t10,c12 CLA, with focus on time point and duration of treatment during adipogenesis. We have shown that t10,c12 CLA treatment has its most attenuating effect early (day (D) 0–6) during differentiation. Treatment during this period is sufficient to prevent lipid accumulation in mature adipocytes. The adipogenic marker genes PPARγ and CCAAT/enhancer binding protein α (C/EBPα) are both down-regulated after treatment within the period from D0–6, while additional treatment also down-regulates the expression of sterol regulatory element binding protein-1c (SREBP-1c), liver X receptor α (LXRα), fatty acid binding protein (aP2), fatty acid translocase (CD36) and insulin-sensitive glucose transporter 4 (GLUT4). These effects of t10,c12 CLA reflect the subsequent attenuation of lipid accumulation observed in mature adipocytes. Interestingly, the early B-cell factor (O/E-1), which is known to promote adipogenesis and to be involved in control of genes important for terminal adipocyte differentiation, is unaffected by treatment of t10,c12 CLA. Taken together, our data indicate that inhibition of lipid accumulation induced by t10,c12 CLA treatment during adipocyte differentiation is associated with a tight regulatory cross-talk between early (PPARγ and C/EBPα) and late (LXRα, aP2 and CD36) adipogenic marker genes.
Keywords: Peroxisome proliferator-activated receptor γ (PPARγ); Liver X receptor α (LXRα); Conjugated linoleic acid; Early B-cell factor (O/E-1); Insulin-sensitive glucose transporter 4 (GLUT4); Adipocyte;
Effects of structural changes of fatty acids on lipid accumulation in adipocytes and primary hepatocytes by Linda Granlund; Laila N. Larsen; Hilde I. Nebb; Jan I. Pedersen (23-30).
Conjugated linoleic acids (CLAs), tetradecylthioacetic acid (TTA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are all shown to differently affect lipid homeostasis. Additionally, previous studies have shown that introducing a methyl group in the molecule potentiates the hypolipidemic effect of EPA. The objective of this study was to determine how cis9,trans11 CLA, trans10,cis12 CLA, TTA, EPA and DHA affect lipid accumulation in 3T3-L1 adipocytes and in cultured primary rat hepatocytes, and to what extent changes in cis/trans configuration or introducing a methyl group in the molecules influence their way of affecting lipid accumulation in these cells. Our results show that trans10,cis12 CLA is highly specific in preventing lipid accumulation in adipocytes, and that small structural changes in the molecule (changing to trans/trans or introducing an α-methyl group) totally abolish this effect and up-regulate the expression levels of adipogenic marker genes towards control levels. Furthermore, all the fatty acids increased hepatic lipid accumulation, whereas the lipid content was normalized after adding an α-methyl group into the molecules. Taken together, our data demonstrate that the various fatty acids are highly specialized molecules, and that small structural changes markedly alter their way of affecting lipid accumulation in adipocytes and hepatocytes.
Keywords: Adipocyte; Peroxisome proliferator-activated receptor (PPAR); Liver X receptor (LXR); Conjugated linoleic acid (CLA); Tetradecylthioacetic acid (TTA); α-Methyl derivative;
Characterization of a ceramide kinase-like protein by Frédéric Bornancin; Diana Mechtcheriakova; Samantha Stora; Christine Graf; Alexander Wlachos; Piroska Dévay; Nicole Urtz; Thomas Baumruker; Andreas Billich (31-43).
Ceramide is a key player governing cell fate, and its conversion to ceramide-1-phosphate by ceramide kinase (CERK) is emerging as an important mean to regulate apoptosis and inflammatory processes. We identified a new ceramide kinase homolog, designated CERK-like protein (CERKL) and we compared it to the known CERK. Real time-PCR analysis of human tissues revealed a restricted pattern of expression for CERKL mRNA. Surprisingly, various ceramides, known substrates for CERK, were not phosphorylated by CERKL in vitro. Upon 32Pi-pulse labeling of COS-1 cells transiently expressing CERKL, or incubation with NBD-C6-ceramide, ceramide-1-phosphate was not detected. After recombinant expression in COS-1 cells, CERKL was partially recovered in the soluble fraction, as a phosphorylated protein. Live cell imaging indicated localization of GFP-tagged CERKL to many cell compartments, including specific association with nucleoli. Two splice variants of CERKL did not localize to nucleoli nor did a CERKL variant with a point mutation in the putative ATP binding site. We also studied a naturally occurring CERKL mutant (R257X), recently linked to the pathology of retinitis pigmentosa. It accumulated in the nucleus but was not associated with nucleoli. Treatment with the calcium ionophore A23187 led to clearing of CERKL from nucleoli, but had no effect on the R257X CERKL mutant. Collectively, although kinase activity of CERKL remains to be proven, these findings suggest a functional link between CERKL and its nucleolar localization. Furthermore, we propose that the cause for retinitis pigmentosa in patients bearing the CERKL R257X mutation might be the accumulation of a truncated CERKL protein in the nucleus.
Keywords: Ceramide kinase; Ceramide-1-Phosphate; Nucleolus; Retinitis pigmentosa;
Regulation of sterol 27-hydroxylase in human monocyte-derived macrophages: up-regulation by transforming growth factor β1 by Magnus Hansson; Kjell Wikvall; Amir Babiker (44-51).
Regulatory mechanisms for human CYP27A1 enzyme have not yet been fully investigated. Our approach was to add different hormones and cytokines to cultured human monocyte-derived macrophages, and assess the effects on the CYP27A1 by measuring the production of 27-hydroxylated cholesterol in the media. Of the different hormones and cytokines tested, only transforming growth factor β1 (TGF-β1) had a clear effect on CYP27A1. Further experiments showed a significant increase in 27-hydroxylated cholesterol products (27-hydroxycholesterol and 3β-hydroxy-5-cholestenoic acid). A concomitant increase in CYP27A1 mRNA levels was also seen and this positive effect was confirmed using a human CYP27A1 luciferase reporter gene expressed in HepG2 cells. Experiments with progressive deletion/luciferase reporter gene constructs indicated that a TGF-β1 responsive sequence might be localized in a region about 400 bp upstream of the CYP27A1 translation start. The possibility is discussed that induction of CYP27A1 by TGF-β1 may be responsible for some of the anti-atherogenic properties of this cytokine.
Keywords: 27-Hydroxycholesterol; 3β-Hydroxy-5-cholestenoic acid; CYP27A1; Cholesterol;
Chemokines bind to sulfatides as revealed by surface plasmon resonance by Roger Sandhoff; Heike Grieshaber; Roghieh Djafarzadeh; Tjeerd P. Sijmonsma; Amanda E.I. Proudfoot; Tracy M. Handel; Herbert Wiegandt; Peter J. Nelson; Hermann-Josef Gröne (52-63).
Chemokines bind to sulfated cell surface glycosaminoglycans and thereby modulate signaling mediated by G-protein-coupled seven-transmembrane domain chemokine receptors. Similar to glycosaminoglycans, sulfated oligosaccharides are also exposed on the cell surface by sulfatides, a class of glycosphingolipids. We have now identified sulfated glycosphingolipids (sulfatides) as novel binding partners for chemokines. Using surface plasmon resonance (SPR), the binding of proinflammatory and homeostatic chemokines to glycosphingolipids, in particular sulfatides, was investigated. Chemokines were immobilized while glycosphingolipids or additional phospholipids incorporated into liposomes were applied as soluble analytes. A specific affinity of the chemokines MCP-1/CCL2, IL-8/CXCL8, SDF-1α/CXCL12, MIP-1α/CCL3 and MIP-1β/CCL4 to the sulfatides SM4s, SM3, SM2a and SB2, SB1a was detected. No significant interactions with the chemokines were observed for gangliosides, neutral glycosphingolipids or phospholipids. Chemokine receptors have been associated with the detergent-insoluble fraction supposed to contain ‘rafts’, i.e., glycosphingolipid enriched microdomains of the cell surface. Accordingly, the data suggest that early chemokine receptor signaling may take place in the vicinity of sulfated glycosphingolipids on the cell surface, whereby these sulfatides could modulate the chemokine receptor-mediated cell activation signal.
Keywords: Chemokine; Sulfatide; Surface plasmon resonance; Liposome; Glycosphingolipid; Glycosaminoglycan;
Molecular characterization of the lipopolysaccharide/platelet activating factor- and zymosan-induced pathways leading to prostaglandin production in P388D1 macrophages by Ralph H. Schaloske; Jarrod W. Provins; Ursula A. Kessen; Edward A. Dennis (64-75).
P388D1 cells release free arachidonic acid (AA) and prostaglandin E2 (PGE2) upon stimulation with platelet-activating factor (PAF) and zymosan. The response to PAF is dependent on priming of the cells with bacterial lipopolysaccharide (LPS). In the LPS/PAF pathway, both AA and PGE2 release are dependent on transcription and translation, whereas in the zymosan pathway the release of these compounds appears to be largely independent of these processes. Using quantitative real-time PCR, we analyzed the expression of mRNAs that encode proteins potentially responsible for the dependency of the LPS/PAF pathway on gene expression. These include all the phospholipases A2 (PLA2) that we detected in P388D1 cells, cyclooxygenases (COX), COX-1 and COX-2, the membrane-associated prostaglandin E synthase-1 (mPGES-1), the lipocalin-type prostaglandin D2 synthase (PGDS), hematopoietic PGDS and the subunit Gαi2 of heterotrimeric G-proteins. None of the mRNAs encoding PLA2s, PGDSs, or Gαi2 are substantially altered during LPS priming. However, cyclooxygenase-2 is up-regulated during LPS priming and after stimulation of the cells with zymosan. A modest but significant increase of mPGES-1 mRNA was also detected upon stimulation with zymosan. Thus, the dependency of the LPS/PAF-induced PGE2 production on gene expression can be attributed to the production of cyclooxygenase-2. The dependency of AA release on gene expression is not due to altered expression of any of the PLA2s. We suggest that an accessory regulatory protein affecting the release of AA must be responsible. Using HPLC we separated lipids that are secreted upon stimulation with LPS/PAF and zymosan and found that in both pathways PGD2 is the dominant prostaglandin produced and also detected PGE2, PGF2α and AA besides several unidentified compounds.
Keywords: Arachidonic acid; Cyclooxygenase; Macrophage; Phospholipase A2; Prostaglandin; Zymosan;
Atorvastatin improves peroxisome proliferator-activated receptor signaling in cardiac hypertrophy by preventing nuclear factor-κB activation by Anna Planavila; Juan C. Laguna; Manuel Vázquez-Carrera (76-83).
Nuclear factor (NF)-κB signaling pathway plays a pivotal role in cardiac hypertrophy. Although it has been reported that statins inhibit cardiac hypertrophy by reducing generation of reactive oxygen species, it is not yet known whether statins prevent NF-κB activation and whether this effect can be related to the reduction in the peroxisome proliferator-activated receptor (PPAR) pathway. In this study, we examined the role of atorvastatin on NF-κB activity and PPAR signaling in pressure overload-induced cardiac hypertrophy. Our findings indicate that atorvastatin inhibits cardiac hypertrophy and prevents the fall in the protein levels of PPARα and PPARβ/δ. Further, atorvastatin treatment avoided NF-κB activation during cardiac hypertrophy, reducing the protein–protein association between these PPAR subtypes and the p65 subunit of NF-κB. These findings indicate that negative cross-talk between NF-κB and PPARs may interfere with the transactivation capacity of the latter, leading to a fall in the expression of genes involved in fatty acid metabolism, and that these changes are prevented by statin treatment.
Keywords: Hypertrophy; Myocyte; Energy metabolism; Statin;
Novel pathways of bile acid metabolism involving CYP3A4 by Karl Bodin; Ulla Lindbom; Ulf Diczfalusy (84-93).
The hepatic predominating cytochrome P450, CYP3A4, plays an essential role in the detoxification of bile acids and is important in pathological conditions such as cholestasis where CYP3A4 is adaptively up-regulated. However, the mechanism that triggers the up-regulation of CYP3A4 is still not clear. In this study, using recombinant CYP3A4 and human liver microsomes, we demonstrate that CYP3A4 can metabolise lithocholic acid into 3-dehydrolithocholic acid, a potent activator of the nuclear receptors, pregnane X receptor and 1,25-dihydroxy vitamin D3 receptor, which are known to regulate the expression of CYP3A4. This process thus provides a feed-forward metabolism of toxic bile acid that may be of importance in maintaining bile acid homeostasis. We also provide evidence for a novel CYP3A4-mediated metabolic pathway of the secondary bile acid deoxycholic acid. Patients treated with the antiepileptic drug carbamazepine, a CYP3A4 inducer, had markedly elevated urinary excretion of 1β-hydroxydeoxycholic acid compared to healthy controls. The importance of CYP3A4 in this process was verified by incubations with recombinant CYP3A4 and human liver microsomes, both of which efficiently converted deoxycholic acid into 1β-hydroxydeoxycholic acid. Interestingly, CYP3A4 was also found to be active against the secondary bile acid ursodeoxycholic acid.
Keywords: Pregnane X receptor; Cholestasis; 3-Dehydro bile acid; 1β-Hydroxy bile acid; Carbamazepine;
Cloning of alkaline sphingomyelinase from rat intestinal mucosa and adjusting of the hypothetical protein XP_221184 in GenBank by Jun Wu; Yajun Cheng; Carina Palmberg; Tomas Bergman; Åke Nilsson; Rui-Dong Duan (94-102).
Intestinal alkaline sphingomyelinase (alk-SMase) digests sphingomyelin and the process may influence colonic tumorigenesis and cholesterol absorption. We recently identified the gene of human alk-SMase and cloned the cDNA. Cross-species screening of homology in GenBank found a hypothetical rat protein, XP_221184, with 491 amino acid residues, which shares 73% identity with human alk-SMase. Based on the cDNA sequence of this protein, we cloned a cDNA from rat intestinal mucosa by RT-PCR. The cloned cDNA encodes a protein with 439 amino acid residues and higher (85%) identity with human alk-SMase. The cloned cDNA differed from the XP_221184 cDNA in splice sites linking exons 2 and 3, and exons 3 and 4, respectively. In the sequence of the cloned protein, the predicted activity motif, sphingomyelin binding sites, and potential glycosylation sites in human alk-SMase are all conserved. To confirm the cloned protein is the real form of alk-SMase, native alk-SMase was purified from rat intestine and subjected to proteolytic digestion followed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and electrospray ionization (ESI) tandem mass spectrometry. Seven tryptic peptides were found to match the cloned protein sequence. Transient expression of the cloned cDNA linked with a myc tag in COS-7 cells demonstrated high SMase activity, with an optimal pH at 9.0 and a specific dependence on taurocholate and taurochenodeoxycholate. The expressed protein reacted with both anti-myc and anti-human alk-SMase antibodies. Northern blotting of rat tissues revealed high levels of mRNA in jejunum but not in other tissues. In conclusion, we cloned rat alk-SMase cDNA from rat intestine, adjusted the putative rat alk-SMase protein in GenBank, and confirmed the specific expression of the gene in the small intestine.
Keywords: Cloning; Alkaline sphingomyelinase; XP_221184;
Conjugated linoleic acid reduction of murine mammary tumor cell growth through 5-hydroxyeicosatetraenoic acid by Jung-Hyun Kim; Neil E. Hubbard; Vincent Ziboh; Kent L. Erickson (103-109).
Conjugated linoleic acid (CLA) is a dietary fatty acid that has been shown to reduce tumorigenesis and metastasis in breast, prostate and colon cancer in animals. However, the mechanism of its action has not been clarified. The goal of this study was to determine whether CLA altered mouse mammary tumor cell growth and whether specific metabolites of the lipoxygenase pathway were involved in CLA action. Both t10, c12-CLA and a lipoxygenase inhibitor, but not c9, t11-CLA or linoleic acid (LA), reduced mouse mammary tumor cell viability and growth by inducing apoptosis and reducing cell proliferation. t10, c12-CLA reduced the production of the 5-lipoxygenase metabolite, 5-hydroxyeicosatetraenoic acid (5-HETE). That effect was not seen with c9, t11-CLA or LA. Adding 5-HETE back to tumor cells reduced the t10, c12-CLA effect on both apoptosis and cell proliferation. These data suggest that t10, c12-CLA reduction of tumor cell growth may involve the suppression of the 5-lipoxygenase metabolite, 5-HETE, with subsequent effects on apoptosis and cell proliferation.
Keywords: Conjugated linoleic acid; 5-Hydroxyeicosatetraenoic acid; 5-Lipoxygenase; Apoptosis; Proliferation;
A comparative study of the effect of the antineoplastic ether lipid 1-O-octadecyl-2-O-methyl-glycero-3-phosphocholine and some homologous compounds on PKCα and PKCɛ by Pablo Conesa-Zamora; Faustino Mollinedo; Senena Corbalán-García; Juan C. Gómez-Fernández (110-119).
The effects of the anti-neoplastic ether lipid ET-18-OCH3 and some structural homologues on the activity of protein kinase Cα (PKCα) were studied and compared with the effects the same had on the activity of PKCɛ. ET-18-OCH3 progressively inhibited the activity of PKCα as the concentration was increased up to 30 mol% of the total lipid, above which the effect was one of activation. The experiments carried out with the homologues showed that the methoxy group bound at the sn-2 position of the glycerol of ET-18-OCH3 is essential for both the initial inhibitory effect and the subsequent activation effect. On the other hand, variations in the type of bond linking substitutions in the sn-1 position, ether or ester, do not seem to play an important role in determining the activity of the enzyme. The effects were different on PKCɛ since ET-18-OCH3 had a triphasic effect, activating the enzyme at low concentrations, inhibiting it at slightly higher concentrations and then activating it again at higher concentrations. In this case, when the homologues were used, it was observed that the presence of the methoxy group linked to the sn-2 position of glycerol and the type of bond linking substitutions to the sn-1 position were important for activating the enzyme, so that only homologues with ester bonds as LPC and PAPC were able to induce the initial activation step in a way similar to ET-18-OCH3. Substitution of the phosphocholine group of ET-18-OCH3 by phosphoserine led to a greater activation of PKCα, an effect that comes from the Ca2+-phospholipid binding site probably because of the specific interaction of this site with the phosphoserine group. The action of ET-18-OCH3 and its homologues, as demonstrated in this paper, may permit the selective inhibition or activation of PKCα and PKCɛ by using the most suitable range of concentrations.
Keywords: Edelphosine; PKC; Anti-neoplastic lipids; ET-18-OCH3; Apoptosis; Cancer;
Biological activities of conjugated fatty acids: conjugated eicosadienoic (conj. 20:2Δc11,t13/t12,c14), eicosatrienoic (conj. 20:3Δc8,t12,c14), and heneicosadienoic (conj. 21:2Δc12,t14/c13,t15) acids and other metabolites of conjugated linoleic acid by Yeonhwa Park; Jayne M. Storkson; Karen J. Albright; Wei Liu; Michael W. Pariza (120-129).
The elongated form of conjugated linoleic acid (CLA), conjugated eicosadienoic acid (CEA, conj. 20:2Δc11,t13/t12,c14), was generated from CLA by liver microsomal fractions. Subsequent testing showed that dietary CEA significantly reduced body fat, and increased lean mass similar to CLA when compared to controls. CEA also decreased lipoprotein lipase activity and triacylglyceride, and increased glycerol release in 3T3-L1 adipocytes, correlated with the trans-12,cis-14 isomer, but CEA required a longer incubation period than cells treated with CLA. Based on the fact that CEA fed animals had CLA in tissue, we suggest that the effect of CEA is due to the CLA converted from CEA in the system. The delta-6 desaturated and elongated form of trans-10,cis-12 CLA (conjugated eicosatrienoic acid, CETA, conj. 20:3Δc8,t12,c14) inhibited LPL activity and increased glycerol release but was less active than trans-10,cis-12 CLA or CEA. The 21-carbon conjugated fatty acid, conjugated heneicosadienoic acid (CHDA, conj. 21:2Δc12,t14/c13,t15), was not active on LPL inhibition, triacylglyceride, or glycerol release in 3T3-L1 adipocytes. We also provide evidence that CLA was metabolized to conjugated dodecadienoic acid (conj. 12:2Δc3,t5/t4,c6). In addition, there were indications of the presence of conjugated tetradecadienoic acid (conj. 14:2Δc5,t7/t6,c8), suggesting that CLA can be metabolized through fatty acid β-oxidation. This is the first work to report the presence of conjugated 12 and 14 carbon fatty acids, originated from CLA, and the biological activities of CEA, CETA and CHDA.
Keywords: Conjugated fatty acid; CLA; Conjugated linoleic acid; Body composition; Lipoprotein lipase; 3T3-L1 cell;
Multiple lipid transport pathways to the plasma membrane in yeast by Martina Schnabl; Günther Daum; Harald Pichler (130-140).
The plasma membrane of the yeast Saccharomyces cerevisiae is devoid of lipid-synthesizing enzymes, but contains all classes of bilayer-forming lipids. As the lipid composition of the plasma membrane does not match any of the intracellular membranes, specific trafficking of lipids from internal membranes, especially the endoplasmic reticulum and the Golgi, to the cell periphery is required. Although the secretory pathway is an obvious route to translocate glycerophospholipids, sphingolipids and sterols to the plasma membrane, experimental evidence for the role of this pathway in lipid transport is rare. Addressing this issue in a systematic way, we labeled temperature-sensitive secretory yeast mutants (sec mutants) with appropriate lipid precursors, isolated the plasma membranes at high purity and quantified labeled lipids of this compartment. Shifting sec mutants to the restrictive temperature reduced transport of both proteins and lipids to the plasma membrane, indicating that the latter compounds are also trafficked to the cell periphery through the protein secretory pathway. However, efficient sec blocks did not abrogate protein and lipid transport, suggesting that parallel pathway(s) for the translocation of membrane components to the plasma membrane of yeast must exist.
Keywords: Yeast; Lipid transport; Secretion; Plasma membrane; Phospholipid; Sterol;
High stearoyl-CoA desaturase protein and activity levels in simian virus 40 transformed-human lung fibroblasts by Natalia Scaglia; J. Matías Caviglia; R. Ariel Igal (141-151).
The precise role of monounsaturated fatty acid (MUFA) synthesis in cell proliferation and programmed cell death remains unknown. The strong correlation of high levels of MUFA and neoplastic phenotype suggest that the regulation of stearoyl CoA desaturase (SCD) must play a significant role in cancer development. In this study, the levels of SCD protein and activity were investigated in normal (WI38) and SV40-transformed (SV40-WI38) human lung fibroblasts. Thus, the activity of SCD on exogenous [14C]stearic acid and endogenous [14C]acetate-labeled fatty acids was increased by 2.2- and 2.6-fold, respectively, in SV40-WI38 compared to WI38 fibroblasts. Concomitantly, a 3.3-fold increase in SCD protein content was observed in SV40-transformed cells. Cell transformation also led to high levels of MUFA, which was paralleled by a more fluid membrane environment. Furthermore, the levels of PPAR-γ, a well-known activator of SCD expression, were highly increased in SV40-transformed fibroblasts. SCD activity appeared linked to the events of programmed cell death, since incubations with 40 μM etoposide induced apoptosis in SV40 cells, and led to a decrease in fatty acid synthesis, SCD activity and in MUFA cellular levels. Taken together, these results suggest that SCD protein and activity levels are associated with the events of neoplastic cell transformation and programmed cell death.
Keywords: Δ9-Desaturation; Fatty acid synthesis; Human cell transformation; Apoptosis; Membrane fluidity;
Temporal gene expression of 3-ketoacyl-CoA reductase is different in high and in low erucic acid Brassica napus cultivars during seed development by Juliette Puyaubert; Wilfrid Dieryck; Patricia Costaglioli; Sylvette Chevalier; Annick Breton; René Lessire (152-163).
The membrane-bound acyl-CoA elongase complex is a key enzyme responsible for erucoyl-CoA synthesis. Among the four putative genes encoding the four moieties of this complex in Brassica napus seeds, only one has been characterized, the Bn-fae1 gene, which encodes the 3-ketoacyl-CoA synthase. The genes encoding the other enzymes (3-ketoacyl-CoA reductase, 3-hydroxyacyl-CoA dehydratase and trans-2,3-enoyl-CoA reductase) have not been identified.We cloned two 3-ketoacyl-CoA reductase cDNA isoforms, Bn-kcr1 and Bn-kcr2, from B. napus seeds. Their function was identified by heterologous complementation in yeast by restoring elongase activities. The comparison of Bn-kcr mRNA expression in different B. napus tissues showed that the genes were preferentially expressed in seeds and roots.We also investigated the regulation of gene expression in High Erucic Acid Rapeseed (HEAR) and in Low Erucic Acid Rapeseed (LEAR) cultivars during seed development. The co-expression of Bn-fae1 and Bn-kcr observed in HEAR cultivar during seed development was different in LEAR cultivar, suggesting that expression of both genes was directly or indirectly linked.
Keywords: Brassica napus; Lipid synthesis; Acyl-CoA elongase; Very long chain fatty acid; Erucic acid; 3-Ketoacyl-CoA reductase;
Aging reduces glycerol-3-phosphate acyltransferase activity in activated rat splenic T-lymphocytes by Lauren W. Collison; Latha Kannan; Thomas M. Onorato; Jens Knudsen; Dipak Haldar; Christopher A. Jolly (164-172).
T-lymphocyte proliferation declines with age. Phosphatidic acid (PA) is the precursor to all glycerophospholipids, which serve as important membrane structural components and signaling molecules. Therefore, we tested the hypothesis that aged T-lymphocyte proliferation may be reduced, in part, suppressing phosphatidic acid (PA) biosynthesis. We showed, for the first time, that anti-CD3 stimulation in rat splenic T-lymphocytes selectively increased mitochondrial glycerol-3-phosphate acyltransferase (GPAT) activity. GPAT activity could be further increased by the addition of recombinant acyl-CoA binding protein (rACBP), but the amplification of GPAT activity was blunted by aging. This is important because PA is the precursor lipid for phospholipid synthesis and GPAT is the rate-limiting enzyme in PA biosynthesis. The mechanism by which stimulation and rACBP increased GPAT activity may involve phosphorylation since incubating Jurkat T-lymphocyte mitochondria with casein kinase 2 in vitro significantly increased GPAT activity. The data presented here suggest a novel mechanism by which aging may reduce activation-dependent mitochondrial GPAT activity. This age-induced alteration would result in reduced PA biosynthesis and could explain, in part, the diminished phospholipid content of the membrane and subsequent loss of proliferative capacity in the aged T-lymphocyte.
Keywords: T-lymphocyte; Phosphatidic acid; Acyl-CoA binding protein; Mitochondria; Aging;
Oxidized but not acetylated low-density lipoprotein reduces preproinsulin mRNA expression and secretion of insulin from HIT-T15 cells by Fumitaka Okajima; Mikiko Kurihara; Chihaya Ono; Yasushi Nakajima; Kyoko Tanimura; Hitoshi Sugihara; Atsushi Tatsuguchi; Kiyotaka Nakagawa; Teruo Miyazawa; Shinichi Oikawa (173-180).
We examined the effect of oxidized low-density lipoprotein (oxLDL) on the insulin secretion in the culture of HIT-T15 cell line, an islet β-cell line derived from a hamster pancreatic tumor. In order to check the uptake of modified LDL by HIT-T15 cells, we prepared DiI-labeled native LDL (nLDL), acetylated LDL (AcLDL), and oxLDL. After the addition of each LDL into the cultures of HIT-T15 cells, fluorescence microscopic study was done. It was suggested that AcLDL and oxLDL were taken up by HIT-T15 cells, as well as nLDL. mRNA expression of the LDL receptor, CD36, and SR-B1 was detected in HIT-T15 by RT-PCR. The medium insulin level was measured in the culture of HIT-T15 cells with each LDL. oxLDL significantly reduced the insulin secretion stimulated by various concentrations of glucose, the intracellular content of insulin, and the expression of preproinsulin mRNA compared to the control cultures without LDL addition. In contrast, nLDL and AcLDL had no effect on the insulin secretion, the intracellular insulin level, or the expression of preproinsulin mRNA. MTT assay findings (reflecting cell numbers) were not different between cultures with and without LDLs. These results indicated that oxLDL disturbed the insulin metabolism of HIT-T15 cells.
Keywords: Oxidized LDL; Scavenger receptor; Pancreatic beta cell; HIT-T15 cell; Lipid peroxidation; Oxidative stress;
Expression of cFABP and PPAR in trophoblast cells: effect of PPAR ligands on linoleic acid uptake and differentiation by Georges Daoud; Lucie Simoneau; André Masse; Eric Rassart; Julie Lafond (181-194).
Throughout gestation, fetal growth depends, in part, on placental transfer of maternal essential fatty acid (EFA) and long-chain polyunsaturated fatty acid. All fatty acid (FA) can cross lipid bilayer by simple diffusion, such as those in the syncytiotrophoblasts, the multinucleated, terminally differentiated trophoblast cells. The trophoblasts differentiation process is accompanied by an increase of human chorionic gonadotropin (hCG) secretion and an inhibition of Human Achaete-Scute Homologue-2 expression (Hash-2). Furthermore, a number of FA-binding proteins (FABPs) have been identified in membrane and cytoplasm of mammalian cells, which are thought to facilitate the transfer of FA across membranes and their intracellular channeling. Thus, the aim of this study was to investigate the implication of cFABPs in linoleic acid (LA) uptake by human trophoblast cells according to differentiation. Moreover, since peroxisome proliferator-activated receptor (PPARs) regulate the expression of cFABP and play an important role in trophoblast cells differentiation, the effects of PPARs ligands are verified on cFABP expression and differentiation. Herein, we reported the increase of the expression of liver and heart FABP (L- and H-FABP) upon differentiation of trophoblast cells, an inhibition of PPARα and β, while PPARγ levels remains unchanged. The nonselective peroxisome-proliferating agents, bezafibrate and LA, impaired trophoblast differentiation, and reduced L- and H-FABP expression. Furthermore, cobalt, a chemical agent known to mimic hypoxia, inhibits trophoblast cells differentiation and diminishes H-, L-FABP and PPARs expression. Finally, both treatments show no influence on LA uptake by trophoblast cells. In conclusion, this study showed that there is no correlation between the expression of H- and L-FABP and LA uptake by trophoblast cells and that bezafibrate and LA greatly impaired trophoblast cells differentiation.
Keywords: cFABP; PPAR; Hash-2; Trophoblast cell; Linoleic acid; Placenta;
Author Index (195-196).
Cumulative Contents (197-198).