BBA - Molecular and Cell Biology of Lipids (v.1737, #1)

As high telomerase activity is detected in most cancer cells, telomerase represents a promising cancer therapeutic target. We investigated the inhibitory effect of various fatty acids on telomerase, with particular emphasis on those with antitumor properties, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). To evaluate the direct effect of fatty acids on telomerase, cell lysates of DLD-1 human colorectal adenocarcinoma cells were mixed with sample fatty acids, and the telomerase activity was determined. Saturated fatty acids and trans-fatty acids showed very weak or no inhibition of telomerase. In contrast, cis-unsaturated fatty acids significantly inhibited the enzyme, and the inhibitory potency was elevated with an increase in the number of double bonds. Accordingly, polyunsaturated fatty acids (PUFAs), like EPA and DHA, appeared to be powerful telomerase inhibitors. To assess the transcriptional effect, DLD-1 cells were cultured in the presence of sample fatty acids, and telomerase activity and gene expression were subsequently evaluated. Culturing DLD-1 cells with either EPA or DHA resulted in a remarkable decrease in telomerase activity. EPA and DHA inhibited telomerase by down-regulating human telomerase reverse transcriptase (hTERT) and c-myc expression via protein kinase C inhibition. These results indicate that PUFAs can directly inhibit the enzymatic activity of telomerase as well as modulate the telomerase at the transcriptional level.
Keywords: Telomerase inhibition; PUFA; hTERT; c-myc; PKC;

Application of 31P MRS to the analysis of phospholipid changes in plasma of patients with acute leukemia by Małgorzata Kuliszkiewicz-Janus; Mariusz A. Tuz; Stanisław Baczyński (11-15).
The aim of the experiment was to evaluate the changes of phospholipid concentrations in patients (n  = 30) with acute leukemia compared with reference group of healthy volunteers (n  = 21). The analysis focused on the following phospholipids (PL) collected from plasma: phosphatidylcholine (PC), plasmalogen of phosphatidylcholine (CPLAS), lysophosphatidylcholine (LPC), sphingomyelin (SM), phosphatidylethanolamine (PE), and phosphatidylinositol (PI). Phospholipid extracts were obtained by Folch's method from 4 ml of plasma. 31P MR spectra were obtained on an AMX Bruker 300 MHz (7.05 T) spectrometer. Calculation of concentration based on integral intensity of the phospholipid relative to an internal concentration standard of MDPA. For healthy volunteers, the following values of phospholipid concentrations were obtained: (5.18 ± 1.615) mmol/l for PC + CPLAS; (0.364 ± 0.178) mmol/l for LPC; (1.211 ± 0.411) mmol/l for SM; (0.343 ± 0.124) mmol/l for PI + PE. PLs of patients were assayed at least twice: at the time of diagnosis and, when appropriate, at the time of complete remission from the disease (CR). At the time of diagnosis, the mean concentrations of studied compounds were: (1.602 ± 0.716) mmol/l for PC + CPLAS; (0.041 ± 0.048) mmol/l for LPC; (0.398 ± 0.198) mmol/l for SM; (0.045 ± 0.071) mmol/l for PI + PE. After attainment of complete remission (CR), the respective values were as follows: (4.094 ± 1.886) mmol/l for PC + CPLAS; (0.295 ± 0.139) mmol/l for LPC; (1.123 ± 0.634) mmol/l for SM; (0.230 ± 0.125) mmol/l for PI + PE. All concentrations found in patients at the time of diagnosis were significantly lower than in reference group and in those benefited from complete remission (CR). By contrast the differences in concentrations of phospholipids in plasma between patients with complete remission (CR) and healthy volunteers were no statistically significant.
Keywords: Acute leukemia; Phospholipid of plasma; 31P MRS in vitro;

Inhibition of apolipoprotein AI gene expression by 1, 25-dihydroxyvitamin D3 by Kent Wehmeier; Ann Beers; Michael J. Haas; Norman C.W. Wong; Andreas Steinmeyer; Ulrich Zugel; Arshag D. Mooradian (16-26).
Members of the steroid receptor superfamily are known to alter the transcription of apolipoprotein AI (apo AI), the major apoprotein of high-density lipoprotein (HDL). To assess the role of vitamin D receptor (VDR) in apo AI gene expression, we investigated the effect of 1α, 25-dihydroxycholecalciferol (1, 25-(OH)2 D3) as well as the vitamin D antagonist ZK-191784 (ZK), on apo AI gene expression and promoter activity in the human hepatoma cell line HepG2. Apo AI secretion and mRNA levels were both suppressed in a dose-dependent manner in HepG2 cells treated 1, 25-(OH)2 D3. This was accompanied by a similar decrease in apo AI promoter activity. Mapping of the vitamin D response element showed that suppression required a region of the apo AI gene promoter identified previously to contain site A. However, vitamin D treatment had no effect on nuclear factor binding to site A of the apo AI promoter. Treatment with vitamin D receptor antagonist ZK inhibited the ability of 1, 25-(OH)2 D3 to repress apo AI promoter activity, while higher doses of ZK increased apo AI promoter activity. ZK did not alter estradiol stimulated apo AI promoter activity. The VDR antisense ODN had no effect on apo AI promoter activity in control cells, however, it reversed the repression normally seen in cells treated with 1, 25-(OH)2D3. It is concluded that 1, 25-(OH)2 D3 suppresses apo A1 gene expression at the transcriptional level, possibly by altering coactivators or corepressors. This effect requires the VDR as well as a vitamin D response element in the apo AI promoter.
Keywords: 1, 25-Dihydroxyvitamin D3; Apolipoprotein AI; Transcriptional regulation;

Pulmonary surfactant function is abolished by an elevated proportion of cholesterol by Lasantha Gunasekara; Samuel Schürch; W. Michael Schoel; Kaushik Nag; Zoya Leonenko; Michael Haufs; Matthias Amrein (27-35).
A molecular film of pulmonary surfactant strongly reduces the surface tension of the lung epithelium–air interface. Human pulmonary surfactant contains 5–10% cholesterol by mass, among other lipids and surfactant specific proteins. An elevated proportion of cholesterol is found in surfactant, recovered from acutely injured lungs (ALI). The functional role of cholesterol in pulmonary surfactant has remained controversial. Cholesterol is excluded from most pulmonary surfactant replacement formulations, used clinically to treat conditions of surfactant deficiency. This is because cholesterol has been shown in vitro to impair the surface activity of surfactant even at a physiological level. In the current study, the functional role of cholesterol has been re-evaluated using an improved method of evaluating surface activity in vitro, the captive bubble surfactometer (CBS). Cholesterol was added to one of the clinically used therapeutic surfactants, BLES, a bovine lipid extract surfactant, and the surface activity evaluated, including the adsorption rate of the substance to the air–water interface, its ability to produce a surface tension close to zero and the area compression needed to obtain that low surface tension. No differences in the surface activity were found for BLES samples containing either none, 5 or 10% cholesterol by mass with respect to the minimal surface tension. Our findings therefore suggest that the earlier-described deleterious effects of physiological amounts of cholesterol are related to the experimental methodology. However, at 20%, cholesterol effectively abolished surfactant function and a surface tension below 15 mN/m was not obtained. Inhibition of surface activity by cholesterol may therefore partially or fully explain the impaired lung function in the case of ALI. We discuss a molecular mechanism that could explain why cholesterol does not prevent low surface tension of surfactant films at physiological levels but abolishes surfactant function at higher levels.
Keywords: Captive bubble surfactometer; Atomic force microscopy; Pulmonary surfactant; Cholesterol; Dipalmitoylphosphatidylcholine (DPPC); Bovine Lipid Extract Surfactant (BLES);

Octanoate reduces very low-density lipoprotein secretion by decreasing the synthesis of apolipoprotein B in primary cultures of chicken hepatocytes by Shizuko Tachibana; Kan Sato; Yoshitake Cho; Tomoyuki Chiba; Wolfgang J. Schneider; Yukio Akiba (36-43).
Fatty acids of varying lengths and saturation differentially affect plasma apolipoprotein B (apoB) levels. To identify the mechanisms underlying the effect of octanoate on very low-density lipoprotein (VLDL) secretion, chicken primary hepatocytes were incubated with either fatty acid-bovine serum albumin (BSA) complexes or BSA alone. Addition of octanoate to culture medium significantly reduced VLDL-triacylglycerol (TG), VLDL-cholesterol and apoB secretion from hepatocytes compared to both control cultures with BSA only and palmitate treatments, but did not modulate intracellular TG accumulation. However, no differences in cellular microsomal triglyceride transfer protein levels were observed in the cultures with saturated fatty acid. In pulse-chase studies, octanoate treatment resulted in reduced apoB-100 synthesis, in agreement with its promotion of secretion. This characteristic effect of octanoate was confirmed by addition of a protease inhibitor, N-acetyl-leucyl-leucyl-norleucinal (ALLN), to hepatocyte cultures. Analysis showed that the level of apoB mRNA was lower in cultures supplemented with octanoate than in the control cultures, but no significant changes were observed in the levels of apolipoprotein A-I, fatty acid synthase and 3-hydroxy-3-methylglutaryl-CoA reductase mRNA as a result of octanoate treatment. Time-course studies indicate that a 50% reduction in apoB mRNA levels requires 12 h of incubation with octanoate. We conclude that octanoate reduced VLDL secretion by the specific down-regulation of apoB gene expression and impairment of subsequent synthesis of apoB, not by the modulation of intracellular apoB degradation, which is known to be a major regulatory target of VLDL secretion of other fatty acids.
Keywords: Apolipoprotein B; Chicken hepatocyte primary culture; Octanoate; Very low density lipoprotein;

Serine palmitoyl-CoA transferase (SPT) deficiency and sphingolipid levels in mice by Mohammad Reza Hojjati; Zhiqiang Li; Xian-Cheng Jiang (44-51).
Sphingolipids play a very important role in cell membrane formation, signal transduction, and plasma lipoprotein metabolism, and all these functions may have an impact on atherosclerotic development. Serine palmitoyl-CoA transferase (SPT) is the key enzyme in sphingolipid biosynthesis. To evaluate in vivo SPT activity and its role in sphingolipid metabolism, we applied homologous recombination to embryonic stem cells, producing mice with long chain base 1 (Sptlc1) and long chain base 2 (Sptlc2), two subunits of SPT, gene deficiency. Homozygous Sptlc11 and Sptlc2 mice are embryonic lethal, whereas heterozygous versions of both animals (Sptlc1+/−, Sptlc2+/−) are healthy. Analysis showed that, compared with WT mice, Sptlc1+/− and Sptlc2+/− mice had: (1) decreased liver Sptlc1 and Sptlc2 mRNA by 44% and 57% (P  < 0.01 and P  < 0.0001, respectively); (2) decreased liver Sptlc1 mass by 50% and Sptlc2 mass by 70% (P  < 0.01 and P  < 0.01, respectively), moreover, Sptlc1 mass decreased by 70% in Sptlc2+/− mouse liver, while Sptlc2 mass decreased by 53% in Sptlc1+/− mouse liver (P  < 0.001 and P  < 0.01, respectively); (3) decreased liver SPT activity by 45% and 60% (P  < 0.01, respectively); (4) decreased liver ceramide (22% and 39%, P  < 0.05 and P  < 0.01, respectively) and sphingosine levels (22% and 31%, P  < 0.05 and P  < 0.01, respectively); (5) decreased plasma ceramide (45% and 39%, P  < 0.01, respectively), sphingosine-1-phosphate (31% and 32%, P  < 0.01, respectively) and sphingosine levels (22.5% and 25%, P  < 0.01, respectively); (6) dramatically decreased plasma lysosphingomyelin (17-fold and 16-fold, P  < 0.0001, respectively); and (7) no change of plasma sphingomyelin, triglyceride, total cholesterol, phospholipids, and liver sphingomyelin levels. These results indicated that both Sptlc1 and Sptlc2 interactions are necessary for SPT activity in vivo, and that SPT activity directly influences plasma sphingolipid levels. Furthermore, manipulation of SPT activity might well influence the course of such diseases as atherosclerosis.

Conjugated linoleic acid (CLA) induces a body fat loss that is enhanced in mice fed coconut oil (CO), which lacks essential fatty acids (EFA). Our objective was to determine if CO enhancement of CLA-induced body fat loss is due to the lack of EFA. The CLA–EFA interaction was tested by feeding CO and fat free (FF) diets for varying times with and without replenishment of individual EFA. Mice fed CO during only the 2-week CLA-feeding period did not differ from control mice in their adipose EFA content but still tended (P  = 0.06) to be leaner than mice fed soy oil (SO). Mice raised on CO or FF diets and fed CLA were leaner than the SO + CLA-fed mice (P  < 0.01). Mice raised on CO and then replenished with linoleic, linolenic, or arachidonic acid were leaner when fed CLA than mice raised on SO (P  < 0.001). Body fat of CO + CLA-fed mice was not affected by EFA addition. In summary, CO-fed mice not lacking in tissue EFA responded more to CLA than SO-fed mice. Also, EFA addition to CO diets did not alter the enhanced response to CLA. Therefore, the increased response to CLA in mice raised on CO or FF diets appears to be independent of a dietary EFA deficiency.
Keywords: Conjugated linoleic acid; Coconut oil; Essential fatty acid; Body fat; Mouse;

Liver cancer is one of the most common cancers worldwide. Altered lipid metabolism in the liver is a key feature of developing liver nodules and tumors. Methods of analysis vary from the most sophisticated chromatography to the in vivo nuclear magnetic resonance (NMR) spectroscopy. In this study, we present a systematic method for the identification and quantitation of signature signals from lipid metabolites using 1D NMR proton spectroscopy. We assessed lipid metabolites in an epigenetic rat hepatocarcinogenesis model induced by treatment with a choline-deficient diet (CDAA, choline-deficient l-amino acid defined) over a period of 1 year, from the formation of steatosis, to the development of nodules and adenomas. A comparable choline-sufficient (CSAA) diet was used for the controls. The resonances of the methylene protons of the glycerol backbone in phospholipids were used to quantify the total concentration of such compounds. CDAA rat livers were found to have significantly higher levels of phospholipids, when compared to CSAA, throughout the entire carcinogenesis period. The tri-methyl protons of choline compounds serves to quantify total choline, and the vinyl and bis-allyl proton resonances can be used to not only quantify fatty acid concentrations but also to probe the number of double bonds in a fatty acid moiety. Early stages of carcinogenesis indicate a lower degree of double bonds in fatty acyl containing compounds in CDAA rat livers, when compared to CSAA. The results of this study are in agreement with those previously published in the literature on other rat hepatocarcinogenesis models.
Keywords: 1H-NMR Spectroscopy; Hepatocarcinogenesis; Rat; Lipid; In vivo;

Novel variants in human and monkey CETP by David B. Lloyd; Jennifer M. Reynolds; Melissa T. Cronan; Suzanne P. Williams; Maruja E. Lira; Linda S. Wood; Delvin R. Knight; John F. Thompson (69-75).
Variation in CETP has been shown to play an important role in HDL-C levels and cardiovascular disease. To better characterize this variation, the promoter and exonic DNA for CETP was resequenced in 189 individuals with extreme HDL-C or age. Two novel amino acid variants were found in humans (V-12D and Y361C) and an additional variant (R137W) not previously studied in vitro were expressed. D-12 was not secreted and had no detectable activity in cells. C361 and W137 retained near normal amounts of cholesteryl ester transfer activity when purified but were less well secreted than wild type. Torcetrapib, a CETP inhibitor in clinical development with atorvastatin, was found to have a uniform effect on inhibition of wild type CETP versus W137 or C361. In addition, the level of variation in other species was assessed by resequencing DNA from nine cynomolgus monkeys. Numerous intronic and silent SNPs were found as well as two variable amino acids. The amino acid altering SNPs were genotyped in 29 monkeys and not found to be significantly associated with HDL-C levels. Three SNPs found in monkeys were identical to three found in humans with these SNPs all occurring at CpG sites.
Keywords: HDL-C; Pharmacogenomics; SNP; CpG;

Acyl coenzyme A dependent retinol esterification by acyl coenzyme A:diacylglycerol acyltransferase 1 by Michael D. Orland; Kamran Anwar; Debra Cromley; Ching-Hsuen Chu; Luping Chen; Jeffrey T. Billheimer; M. Mahmood Hussain; Dong Cheng (76-82).
We provide biochemical evidence that enzymes involved in the synthesis of triacylglycerol, namely acyl coenzyme A:diacylglycerol acyltransferase (DGAT) and acyl coenzyme A:monoacylglycerol acyltransferase (MGAT), are capable of carrying out the acyl coenzyme A:retinol acyltransferase (ARAT) reaction. Among them, DGAT1 appears to have the highest specific activity. The apparent K m values of recombinant DGAT1/ARAT for retinol and palmitoyl coenzyme A were determined to be 25.9 ± 2.1 μM and 13.9 ± 0.3 μM, respectively, both of which are similar to the values previously determined for ARAT in native tissues. A novel selective DGAT1 inhibitor, XP620, inhibits recombinant DGAT1/ARAT at the retinol recognition site. In the differentiated Caco-2 cell membranes, XP620 inhibits ∼85% of the Caco-2/ARAT activity indicating that DGAT1/ARAT may be the major source of ARAT activity in these cells. Of the two most abundant fatty acyl retinyl esters present in the intact differentiated Caco-2 cells, XP620 selectively inhibits retinyl–oleate formation without influencing the retinyl–palmitate formation. Using this inhibitor, we estimate that ∼64% of total retinyl ester formation occurs via DGAT1/ARAT. These studies suggest that DGAT1/ARAT is the major enzyme involved in retinyl ester synthesis in Caco-2 cells.
Keywords: DGAT; ARAT; Retinol; Retinyl ester; DGAT inhibitor;