BBA - Molecular and Cell Biology of Lipids (v.1734, #2)

Herbivore-induced, indirect plant defences by Gen-ichiro Arimura; Christian Kost; Wilhelm Boland (91-111).
Indirect responses are defensive strategies by which plants attract natural enemies of their herbivores that act as plant defending agents. Such defences can be either constitutively expressed or induced by the combined action of mechanical damage and low- or high-molecular-weight elicitors from the attacking herbivore. Here, we focus on two induced indirect defences, namely the de novo production of volatiles and the secretion of extrafloral nectar, which both mediate interactions with organisms from higher trophic levels (i.e., parasitoids or carnivores). We give an overview on elicitors, early signals, and signal transduction resulting in a complex regulation of indirect defences and discuss effects of cross-talks between the signalling pathways (synergistic and antagonistic effects). In the light of recent findings, we review molecular and genetic aspects of the biosynthesis of herbivore-induced plant volatiles comprising terpenoids, aromatic compounds, and metabolites of fatty acids which act as infochemicals for animals and some of which even induce defence genes in neighbouring plants. Finally, ecological aspects of these two indirect defences such as their variability, specificity, evolution as well as their ecological relevance in nature are discussed.
Keywords: Extrafloral nectar; Herbivore-induced plant volatile; Indirect defence; Oxylipin; Signalling pathway;

Fatty acid metabolism is enhanced in type 2 diabetic hearts by Andrew N. Carley; David L. Severson (112-126).
The metabolic phenotype of hearts has been investigated using rodent models of type 2 diabetes which exhibit obesity and insulin resistance: db/db and ob/ob mice, and Zucker fatty and ZDF rats. In general, cardiac fatty acid (FA) utilization is enhanced in type 2 diabetic hearts, with increased rates of FA oxidation (db/db, ob/ob and ZDF models) and increased FA esterification into cellular triacylglycerols (db/db hearts). Hearts from db/db and ob/ob mice and ZDF rat hearts all have elevated levels of myocardial triacylglycerols, consistent with enhanced FA utilization. A number of mechanisms may be responsible for enhanced FA utilization in type 2 diabetic hearts: (i) increased FA uptake into cardiac myocytes and into mitochondria; (ii) altered mitochondrial function, with up-regulation of uncoupling proteins; and (iii) stimulation of peroxisome proliferator-activated receptor-α. Enhanced cardiac FA utilization in rodent type 2 diabetic models is associated with reduced cardiac contractile function, perhaps as a consequence of lipotoxicity and/or reduced cardiac efficiency. Similar results have been obtained with human type 2 diabetic hearts, suggesting that pharmacological interventions that can reduce cardiac FA utilization may have beneficial effects on contractile function.
Keywords: Fatty acid metabolism; Diabetes and cardiac metabolism;

Changes in the expression patterns of genes involved in bile acid (BA) synthesis were investigated during rat liver regeneration that follows two-thirds partial hepatectomy. BAs in bile were measured by GC-MS and the absolute and relative abundance of specific mRNAs in the liver by RT-real-time quantitative PCR. Cyclin E mRNA, used as an indicator of liver cell proliferation, peaked at day 1. The levels of mRNA of α-fetoprotein transcription factor (FTF) and small heterodimer partner (SHP) were first reduced (day 1) and then (days 2–3) increased, when those of farnesoid X receptor (FXR) were also transiently enhanced. The early (day 1) up-regulation of Cyp7a1, and Cyp8b1, together with the down-regulation of Cyp27, was consistent with an increased proportion of cholic acid versus chenodeoxycholic acid and a progressive recovery in total BAs secretion. The transient appearance of flat BAs (allo-BAs plus Δ4-unsaturated-BAs) during rat liver regeneration was probably due to the changes in the expression ratio of steroid 5α- versus 5β-reductase. Both were first (day 1) down-regulated and then up-regulated (5α-reductase more than 5β-reductase). In conclusion, changes in the expression patterns of nuclear receptors and enzymes involved in BA synthesis are consistent with the transient modifications that occur in BA pool during rat liver regeneration.
Keywords: Allo-cholic acid; Cholesterol; Hepatectomy; Metabolism; Nuclear receptor; Proliferation;

mRNA transcripts encoding multiple proteins from the cholesterol biosynthetic and uptake pathways in livers are controlled by sterol regulatory element binding protein-2 (SREBP-2), a membrane-bound mammalian transcription factor. The aims of the present study were to investigate whether SREBP-2 responds to plasma cholesterol levels and modulates expression of factors involved in the cholesterol metabolism of chickens. Supplementing the diets of chickens with 0.1% pravastatin, a drug used to control hypercholesterolemia, decreased plasma LDL-cholesterol concentrations. It also increased levels of the nuclear forms of SREBP-2 and increased gene expression of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) and low-density lipoprotein receptor (LDLr) in livers, relative to a control group. In contrast, feeding of 3% cholesterol-supplemented diet increased plasma total- and LDL-cholesterol concentrations but decreased levels of nuclear forms of SREBP-2 and reduced gene expression of HMGR and LDLr. However, the LDL-binding activities of chicken liver membranes were not affected by plasma cholesterol concentrations or by hepatic levels of the nuclear form of SREBP-2. LDL-binding proteins were detected as bands of 90 and 515 kDa on a ligand blot and the intensity of these bands was unaffected by pravastatin and cholesterol supplementation. These findings suggest that the cholesterol biosynthetic pathway is regulated by the nuclear form of SREBP-2 in chickens as well as in mammals, but that there may be species-specific differences in the regulatory mechanisms of hepatic cholesterol uptake.
Keywords: Sterol regulatory element binding protein-2 (SREBP-2); 3-hydroxy-3-methylglutary coenzyme A reductase (HMGR); LDL receptor (LDLr); Chicken;

Interleukin-6 genotype is associated with high-density lipoprotein cholesterol responses to exercise training by Amy Halverstadt; Dana A. Phares; Stephen Roth; Robert E. Ferrell; Andrew P. Goldberg; James M. Hagberg (143-151).
High-density lipoprotein cholesterol (HDL-C) and its subfractions are modifiable with exercise training and these responses are heritable. The interleukin-6 (IL6)-174G/C polymorphism may be associated with HDL-C levels. We hypothesized that the IL6-174G/C polymorphism would be associated with plasma HDL-C response to exercise training.Sixty-five 50- to 75-year-olds on a standardized diet were studied before and after 24 weeks of aerobic exercise training. Significant differences existed among genotype groups for change with exercise training in HDL-C, HDL3-C, integrated HDL4,5NMR-C, and HDLsize. The CC genotype group increased HDL-C more than the GG (7.0 ± 1.3 v. 1.0 ± 1.1 mg/dL, p = 0.001) and GC groups (3.3 ± 0.9 mg/dL, p = 0.02); for HDL3-C, the CC group increased more than the GG (6.1 ± 1.0 v. 0.9 ± 0.9, mg/dL p < 0.001) and GC groups (2.5 ± 0.7 mg/dL, p = 0.006). Integrated HDL4,5NMR-C increased more in the CC than GG group (6.5 ± 1.6 mg/dL v. 1.0 ± 1.3 mg/dL, p = 0.01), as did HDLsize compared to the GG (CC: 0.3 ± 0.1 v. GG: 0.1 ± 0.1 nm, p = 0.02) and GC (0.0 ± 0.0 nm, p = 0.007) groups.IL6 genotype is associated with HDL-C response to exercise training.
Keywords: Lipid; Lipoprotein; Genetic; Exercise; Risk factor;

Partial truncation of the NH2-terminus affects physical characteristics and membrane binding, aggregation, and fusion properties of annexin A7 by Devendra G. Naidu; Abhijit Raha; Xiao-Liang Chen; Alan R. Spitzer; Avinash Chander (152-168).
Annexin A7 (synexin, annexin VII), a member of the annexin family of proteins, causes aggregation of membranes in a Ca2+-dependent manner and has been suggested to promote membrane fusion during exocytosis of lung surfactant, catecholamines, and insulin. Although annexin A7 (A7) was one of the first annexin proteins described, limited studies of its physical characteristics or of structural domains affecting any of its proposed functions have been conducted. As postulated for other annexin proteins, the unique NH2-domain possibly determines the functional specificity of A7. Therefore, we evaluated the effects of segmental deletions in the NH2-terminus on several characteristics associated with the COOH-terminus of A7. The COOH-terminus contains the only tryptophan residue, and all potential trypsin sites, and the Ca2+ and phospholipid binding sites. Recombinant rat A7 and its deletion mutants were expressed using constructs based on the cDNA sequence obtained by screening a rat lung cDNA library. Ca2+ increased the tryptophan fluorescence of A7 and caused a small red shift in the emission maximum (λmax), which was further increased in presence of phospholipid vesicles (PLV). NH2-terminal deletions of 29, 51, and 109 residues affected the peak width of fluorescence and λmax, surface-exposure of tryptophan residue, and caused a smaller Ca2+-dependent red shift in λmax of membrane-bound protein in comparison to A7. Limited proteolysis with trypsin showed that Ca2+ increased the proteolysis of all proteins, but the deletions also affected the pattern of proteolysis. The presence of PLV protected against Ca2+-dependent increase in proteolysis of all proteins. The deletion of first 29 residues also caused decreased membrane binding, aggregation, and fusion, when compared with A7. Collectively, these results suggest that specific NH2-terminus domains can alter those properties of A7 that are normally associated with the COOH-terminus. We speculate that interactions between the NH2- and COOH-termini are required for membrane binding, and aggregation and fusion properties of annexin A7.
Keywords: Synexin; Annexin VII; Tryptophan fluorescence; Vesicle aggregation; Membrane fusion; Stern-Volmer quenching constant; Deletion mutants; Limited proteolysis;

The degradation of fatty acids having cis- or trans-unsaturated bond at an even carbon was analyzed in Saccharomyces cerevisiae by monitoring polyhydroxyalkanoate production in the peroxisome. Polyhydroxyalkanaote is synthesized by the polymerization of the β-oxidation intermediates 3-hydroxy-acyl-CoAs via a bacterial polyhydroxyalkanoate synthase targeted to the peroxisome. The synthesis of polyhydroxyalkanoate in cells grown in media containing 10-cis-heptadecenoic acid was dependent on the presence of 2,4-dienoyl-CoA reductase activity as well as on Δ32-enoyl-CoA isomerase activity. The synthesis of polyhydroxyalkanoate from 10-trans-heptadecenoic acid in mutants devoid of 2,4-dienoyl-CoA reductase revealed degradation of the trans fatty acid directly via the enoyl-CoA hydratase II activity of the multifunctional enzyme (MFE), although the level of polyhydroxyalkanoate was 10–25% to that of wild type cells. Polyhydroxyalkanoate produced from 10-trans-heptadecenoic acid in wild type cells showed substantial carbon flux through both a reductase-dependent and a direct MFE-dependent pathway. Flux through β-oxidation was more severely reduced in mutants devoid of Δ32-enoyl-CoA isomerase compared to mutants devoid of 2,4-dienoyl-CoA reductase. It is concluded that the intermediate 2-trans,4-trans-dienoyl-CoA is metabolized in vivo in yeast by both the enoyl-CoA hydratase II activity of the multifunctional protein and the 2,4-dienoyl-CoA reductase, and that the synthesis of the intermediate 3-trans-enoyl-CoA in the absence of the Δ32-enoyl-CoA isomerase leads to the blockage of the direct MFE-dependent pathway in vivo.
Keywords: β-Oxidation; Enoyl-CoA isomerase; Dienoyl-CoA reductase; Peroxisome; Polyhydroxyalkanaoate; Saccharomyces cerevisiae;

An elevated plasma level of apolipoprotein B-containing lipoproteins is a risk factor for atherosclerotic cardiovascular disease. Subtle genetic abnormalities in gene expression including an increased expression of the APOB gene may play an important role in determining overall risk. In an attempt to increase mouse Apob expression, we used gene targeting and duplicated ≈65 kb of genomic DNA containing the Apob locus in its natural genomic position in mice. While we successfully generated mice carrying the Apob gene duplication, the amount of the total Apob mRNA was not increased in their liver. In the intestine, total Apob mRNA was reduced to half of the wild-type mice. Plasma lipids in the Apob duplication mice were not altered. Expression analyses showed that the proximal Apob gene in the duplicated locus was preferentially expressed in both tissues suggesting a limitation of tissue-specific enhancer function. The previously characterized distant intestinal control element was not duplicated, explaining the unequal ratio of intestinal Apob expression. While the existence of an additional liver-specific enhancer element is unknown, our findings suggest the presence of an additional enhancer outside the duplicated region, and that Apob gene expression is more complicated than previously thought.
Keywords: Gene duplication; Intestinal control element; Neo gene; Gene targeted gap repair; Apolipoprotein B;

The fungus Aspergillus tamarii transforms progesterone to testololactone in high yield through a flexible four-step enzymatic pathway. To date no studies have investigated the effect of transposition of steroidal functionality between ring-A and ring-D in order to determine the effect on steroidal metabolism. A series of novel quasi reverse steroids (7–9) were synthesised through Linz and Schäfer oxidation where 14-en-16-one functionality is generated on ring-D of the steroid. To retain parity with the normal series ring-D functionality was substituted onto ring-A of the analogues. All of the analogues (7–9) were handled through a minor 11β-hydroxylation pathway with no lactones being formed. In previous studies testololactone is produced within the first 12 h of metabolism. A time course experiment demonstrated that the transformation of these steroids initiated with the formation of a 3β-hydroxy group after which (48–96 h) hydroxylation through a minor pathway occurred, indicating that this hydroxylase was only then being induced. This is in contrast to the normal fungal metabolism of xenobiotic steroidal substrates where they are primarily hydroxylated. Furthermore, ring-D hydrogenation is reported for the first time as is reverse metabolism on this pathway. All metabolites were isolated by column chromatography and were identified by 1H and 13C NMR spectroscopy, DEPT analysis and other spectroscopic and crystallographic data.
Keywords: Aspergillus tamarii; Aflatoxin; Biotransformation; Hydroxylation; Lactonisation; Metabolic pathway;

Leptin inhibits apolipoprotein M transcription and secretion in human hepatoma cell line, HepG2 cells by Guanghua Luo; Maria Hurtig; Xiaoying Zhang; Peter Nilsson-Ehle; Ning Xu (198-202).
Apolipoprotein M (apoM) is a novel apolipoprotein presented mostly in high-density lipoprotein (HDL) in human plasma. Previously we have reported that both leptin and leptin receptor are essential for apoM expression in vivo. The expression of apoM is lower in the leptin deficient (ob/ob) mouse and leptin receptor deficient (db/db) mouse than in the normal mouse. In the present study, however, we demonstrated that supra-physiological concentrations of recombinant leptin significantly inhibited apoM transcription and secretion in the human hepatoma cell line, HepG2 cells. Both Northern blotting and real-time RT-PCR were applied into the analyses of apoM mRNA levels, and compatible data were obtained. The inhibitory effect of leptin on apoM mRNA levels in HepG2 cells is dose dependent, i.e. 100 ng/mL of leptin decreased apoM mRNA levels by 30%, and 500 ng/mL of leptin decreased apoM mRNA levels about 50%. Even at a physiological concentration of leptin (10 ng/mL), apoM expression was decreased, and in parallel, the secretion of apoM into the medium was also decreased. Furthermore, we examined apoAI, apoB and apoE by Northern blotting analyses. The results demonstrated that leptin does not significantly influence the expressions of apoAI, apoB and apoE in HepC2 cells, suggesting that leptin has a specific regulatory effect on hepatic apoM transcription and secretion in vitro. The mechanism on the contradictory effects of leptin on apoM expression in vivo and in vitro needs further investigation.
Keywords: Lipoprotein; Apolipoprotein M; Leptin; HepG2 cell line;

Sterol stringency of proliferation and cell cycle progression in human cells by Yajaira Suárez; Carlos Fernández; Beatriz Ledo; Miguel Martín; Diego Gómez-Coronado; Miguel A. Lasunción (203-213).
Cholesterol is a major component of the plasma membrane in mammalian cells, where it acts as a modulator of bulk physical state and integrity. In addition to its structural role, cholesterol is essential for proliferation and other cell processes. The present study was undertaken to explore the stringency of the requirement for cholesterol as a regulator of proliferation and cell cycle progression. Comparisons were made between cholesterol and other sterol analogs that differ from cholesterol in three specific elements: the presence of a Δ5 double bond in ring B, the hydroxyl group at C-3, and the presence of an aliphatic side chain. The human leukemia cells HL-60 and MOLT-4 were cultured in cholesterol-free medium and treated with different sterols in the presence or absence of SKF 104976, a competitive inhibitor of lanosterol 14α-demethylase that allows the synthesis of isoprenoid derivatives but not cholesterol. Our results show that the β-hydroxyl group at C-3 and the unsaturated bond at Δ5 are necessary for cell proliferation and cell cycle progression. The sterol analog 5α-cholestan-3β-ol (dihydrocholesterol), which is saturated at Δ5 and has an A/B ring junction in the trans configuration, was also able to support cell growth. However, 5β-cholestan-3β-ol and 5β-cholestan-3α-ol, both of which have an A/B ring junction in the cis configuration, were totally ineffective in supporting cell growth. Indeed, they produced an inhibition of cell proliferation and arrested the cell cycle specifically in the G2/M phase. These effects of 5β-cholestanols were abrogated by cholesterol in a concentration-dependent manner. Moreover, 5β-cholestanols potently inhibited cholesterol biosynthesis and transcription driven by the sterol response element. In addition to providing a description of the structural features of sterols associated with their supporting action on cell proliferation in mammalian cells, the present results demonstrate that selected cholesterol analogs may act as cytostatic agents, interrupting cell cycle progression specifically in the G2/M phase.
Keywords: Cell proliferation; Cell cycle; Cholesterol biosynthesis; Cholesterol analog;

Corrigendum to “The role of complex lipids in the synthesis of bioactive aldehydes of the marine diatom Skeletonema costatum” [Biochim. Biophys. Acta 1686 (2004) 100–107] by Giuliana d'Ippolito; Sara Tucci; Adele Cutignano; Giovanna Romano; Guido Cimino; Antonio Miralto; Angelo Fontana (214).