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BBA - Molecular and Cell Biology of Lipids (v.1735, #1)

Editorial Board (pp. ii).

Transcriptional regulatory networks in lipid metabolism control ABCA1 expression by Gerd Schmitz; Thomas Langmann (pp. 1-19).

The ATP-binding cassette transporters, ABCA1 and ABCG1, are major players in mediating cellular efflux of phospholipids and cholesterol to apoA-I containing lipoproteins including preβ-HDL and αHDL and thereby exert important antiatherogenic properties. Although the exact mechanisms how ABC transporters mediate lipid transport are not completely resolved, recent evidence from several laboratories including ours suggests that vesicular transport processes involving different interactive proteins like β2-syntrophin, α1-syntrophin, Lin7, and cdc42 are critically involved in cellular lipid homeostasis controlled by ABCA1 and ABCG1. Besides sterols and fatty acids as known physiological modulators of the LXR/RXR and SREBP pathways, a growing list of natural and synthetic substances and metabolic regulators such as retinoids, PPAR-ligands, hormones, cytokines, and drugs are particularly effective in modulating ABCA1 and ABCG1 gene expression. Although ABCA1 protein amounts are regulated at the level of stability, the majority of potent activating and repressing mechanisms on ABCA1 function directly act on the ABCA1 gene promoter. Among the inducing factors, liver-X-receptors (LXR), retinoic acid receptors (RAR) and peroxisome proliferator-activated receptors (PPARs) along with their coactivators provide an amplification loop for ABCA1 and ABCG1 expression. The ABCA1 promoter is further stimulated by the ubiquitous factor Sp1 and the hypoxia-induced factor 1 (HIF1), which bind to GC-boxes and the E-box, respectively. Shutdown of ABCA1 expression in the absence of sterols or in certain tissues is mediated by corepressor complexes involving unliganded LXR, sterol-regulatory element binding protein 2 (SREBP2), Sp3, and the SCAN-domain protein ZNF202, which also impacts nuclear receptor signaling. Thus, a highly sophisticated transcriptional network controls the balanced expression of ABCA1.

Keywords: Abbreviations; A; _2A; R; adenosine A; _2A; receptor; ABC; ATP-binding cassette; ACAT1; acyl-CoA:cholesterol acyltransferase 1; AP; adapter protein; apo; apolipoprotein; ARL7; ADR-ribosylation factor-like 7; ARNT; arylhydrocarbon receptor nuclear translocator; ATRA; all-trans retinoic acid; CETP; cholesterylester transfer protein; CFTR; cystic fibrosis transmembrane conductance regulator; DMHCA; N; ,; N; -dimethyl-3β-hydroxycholenamide; DR4; direct repeat separated by four nucleotides; ER; estrogen receptor; GGPP; geranylgeranyl pyrophosphate; HAC; histone acetylase; HDAC; histone deacetylase; HDL; high-density lipoprotein; HIF; hypoxia-inducible factor; HP-1; heterochromatin 1; IFN; interferon; IL; interleukin; KRAB; Krüppel associated box; LDL; low-density lipoprotein; LPS; lipopolysaccharide; LXR; liver-X-receptor; PGC; PPARγ coactivator; PKA; protein kinase A; PPAR; peroxisome proliferator activated receptor; RA; retinoic acid; RAR; retinoic acid receptor; RIP140; receptor interacting protein 140; RXR; retinoid-X-receptor; SDP1; SCAN domain protein 1; SMRT; silencing mediator of retinoic acid and thyroid hormone receptors; SRC; steroid receptor coactivator; SREBP; sterol regulatory element binding protein; SUR; sulfonylurea receptor; T3; thyroid hormone; TNF; tumor necrosis factors; TXNIP; thioredoxin-interacting protein; USF; upstream stimulatory factorABCA1; ABCG1; Lipid transport; Nuclear receptor; Sterol regulatory element binding protein; Oxysterols; Retinoid; ZNF202

Efflux of lipid from macrophages after induction of lipid accumulation by chylomicron remnants by Elizabeth H. Moore; Fatos Bejta; Michael Avella; Keith E. Suckling; Kathleen M. Botham (pp. 20-29).

The fate of cholesterol and triacylglycerol taken up and accumulated by macrophages after exposure to chylomicron remnants was investigated using macrophages derived from the human monocyte cell line THP-1 and chylomicron remnant-like particles containing human apolipoprotein (apo) E (CRLPs) as the experimental model. In THP-1 macrophages lipid loaded with CRLPs and incubated with various cholesterol acceptors for 24 h, the mass of cholesterol and cholesteryl ester found in the cells was not changed by HDL, HDL₃ or lipid-free ApoA-I, although it was decreased by 38% by ApoA-I-phosphatidylcholine vesicles (ApoA-I-PC). After loading of the macrophages with [³H]cholesterol-labelled CRLPs, only about 5% of the label was effluxed in 24 h in the absence of cholesterol acceptors, and this increased to about 10% with ApoA-I or PC only, and to about 30% with apoA-I-PC. In similar experiments with [³H]triolein, only about 4% of the labelled triacylglycerol taken up by the cells was released into the medium in 24 h, and a large (>60%) and consistent proportion of the intracellular radioactivity remained associated with the triacylglycerol throughout this period. These results suggest that cholesterol and triacylglycerol derived from chylomicron remnants are not readily cleared from macrophages, and this is likely to contribute to the atherogenicity of the remnant lipoproteins.

Keywords: Chylomicron remnants; Lipid efflux; Cholesterol; Triacylglycerol; Human macrophages

Molecular and cellular effects of cis-9, trans-11-conjugated linoleic acid in enterocytes: Effects on proliferation, differentiation, and gene expression by A. Lampen; M. Leifheit; J. Voss; H. Nau (pp. 30-40).

It has been hypothesized that dietary conjugated linoleic acids (CLA) may inhibit colon tumorigenesis. The aim of our study was to investigate the cellular and molecular effects of cis-9 (9Z), trans-11 (11E)-CLA on the proliferation, differentiation, interaction with peroxisome proliferator-activated receptors (PPARs), and expression of genes relevant in the APC-β-catenin-TCF4 signalling pathway in human HT-29 and Caco-2 colon cells. We found that 9Z,11E-CLA inhibited the proliferation of HT-29 and Caco-2 cells. Trans-vaccenic acid (VA) showed no antiproliferative effects at all. We determined that 9Z,11E-CLA induced cell differentiation as measured by intestinal alkaline phosphatase (IAP) enzyme activity in Caco-2 cells, mRNA expression of IAP, and activation of a 5′ flanking region of IAP. The 9Z,11E-CLA activated human PPARδ as measured in a reporter gene assay. Treatment of HT29 cells in the poliferation phase with 9Z,11E-CLA repressed mRNA-expression of proliferation genes such as c- myc, cyclin D1 and c- jun in a concentration dependent manner. The promoter activities of c- myc and AP1 were also inhibited after incubation with 9Z,11E-CLA. β-Catenin mRNA and protein expression was also repressed by the treatment with 9Z,11E-CLA. In addition, the mRNA expression of PPARδ was repressed by treatment of the HT-29 cells with 9Z,11E-CLA. We conclude that 9Z,11E-CLA has an antiproliferative effect at the cellular and molecular levels in human colon cells. The results indicate that the preventive effects of CLA in the development of colon cancer may be due to their downregulation of some target genes of the APC-β-catenin-TCF-4- and PPARδ signalling pathway.

Keywords: Abbreviations; CLA; conjugated linoleic acid; PPAR; peroxisome proliferator activated receptor; IAP; intestinal alkaline phosphatase; RE; response element; LBD; ligand binding domainCLA; Caco-2; HT29; PPAR; Proliferation; Differentiation

The gene encoding the human ileal bile acid-binding protein (I-BABP) is regulated by peroxisome proliferator-activated receptors by J.F. Landrier; C. Thomas; J. Grober; I. Zaghini; V. Petit; H. Poirier; I. Niot; P. Besnard (pp. 41-49).

Peroxisome proliferator-activator receptors (PPAR) are involved in cholesterol homeostasis through the regulation of bile acids synthesis, composition, and reclamation. As ileal bile acid-binding protein (I-BABP) is thought to play a crucial role in the enterohepatic circulation of bile acids, we investigated whether I-BABP gene expression could also be affected by PPAR. Indeed, treatment with the PPARα-PPARβ/δ agonist bezafibrate led to the up-regulation of I-BABP mRNA levels in the human intestine-derived Caco-2 cells. Cotransfections of the reporter-linked human I-BABP promoter (hI-BABP^−2769/+44) together with PPAR and RXR expression vectors demonstrated that the fibrate-mediated induction of the I-BABP gene is dependent on PPARα or PPARβ/δ. Using progressive 5′ deletions of the hI-BABP promoter and sequence analysis, we identified a putative PPAR-binding site located at the position −198 and −186 upstream of the transcription initiation site. Electrophoretic mobility shift assays showed that the PPAR/RXR heterodimer can specifically bind to this PPRE-like motif. The deletion of the PPRE within the hI-BABP promoter abolished the PPAR-mediated transactivation in transient transfection assays. The regulation of the I-BABP promoter by PPAR appears species-specific, as the mouse I-BABP promoter, which lacks a conserved PPRE, was not responsive to exogenous PPAR expression in the presence of bezafibrate. Our findings show that the I-BABP gene may be a novel target for PPAR in humans and further emphasize the role for PPAR in the control of bile acid homeostasis.

Keywords: Abbreviations; ASBT; apical sodium-dependent bile acid transporter; I-BABP; ileal bile acid-binding protein; CYP7A1; cholesterol 7α-hydroxylase; CYP27A1; sterol 27-hydroxylase; CYP8B1; sterol 12α-hydroxylase; PPAR; peroxisome proliferator-activated receptors; RXR; retinoid-X-receptor; FXR; farnesoid-X-receptor; LXR; liver-X-receptor; SREBP; sterol regulatory element-binding proteins; PPRE; PPAR responsive element; BARE; bile acid responsive element; DR1; direct repeat 1; CAT; chloramphenicol acetyl transferase; CDCA; chenodeoxycholic acid; EMSA; electrophoretic mobility shift assayI-BABP; Bile acid; PPAR; Fibrate

The lipid droplet enzyme Tgl1p hydrolyzes both steryl esters and triglycerides in the yeast, Saccharomyces cerevisiae by Anita Jandrositz; Julia Petschnigg; Robert Zimmermann; Klaus Natter; Hubert Scholze; Albin Hermetter; Sepp D. Kohlwein; Regina Leber (pp. 50-58).

Based on sequence homology to mammalian acid lipases, yeast reading frame YKL140w was predicted to encode a triacylglycerol (TAG) lipase in yeast and was hence named as TGL1, triglyceride lipase 1. A deletion of TGL1, however, resulted in an increase of the cellular steryl ester content. Fluorescently labeled lipid analogs that become covalently linked to the enzyme active site upon catalysis were used to discriminate between the lipase and esterase activities of Tgl1p. Tgl1p preferred single-chain esterase inhibitors over lipase inhibitors in vitro. Under assay conditions optimal for acid lipases, Tgl1p exhibited steryl esterase activity only and lacked any triglyceride lipase activity. In contrast, at pH 7.4, Tgl1p also exhibited TAG lipase activity; however, steryl ester hydrolase activity was still predominant. Tgl1p localized exclusively to lipid droplets which are the intracellular storage compartment of steryl esters and triacylglycerols in the yeast S. cerevisiae. In a tgl1 deletion mutant, the mobilization of steryl esters in vivo was delayed, but not abolished, suggesting the existence of additional enzymes involved in steryl ester mobilization.

Keywords: Abbreviation; LD; lipid droplet; ER; endoplasmic reticulum; TAG; triacylglycerol; FFA; free fatty acid; HLAL; human lysosomal acid lipase; PerGP; 1-; O; -hexadecyl-2-; O; -perylenedodecyl-; sn; -glycero-3-phosphonic acid–(; n; -hexyl)-; p; -nitrophenyl-ester; PerMBP; O; -perylenebutylmethylphosphonic-acid–; p; -nitrophenyl ester; eGFP; enhanced green fluorescent protein; DIC; differential interference contrastLipid droplet; Triacylglycerol lipase; Acid lipase gene family; Fluorescent inhibitor; Steryl ester; Green fluorescent protein

Bile acid induces hydrophobicity-dependent membrane alterations by Sandeep Akare; Jesse D. Martinez (pp. 59-67).

Elevated concentrations of fecal bile acids are a known risk factor for colon cancer, owing to alterations in cellular signaling. In colonic cells, where bile acid uptake is minimal, the hydrophobicity-induced membrane perturbation and alterations have been proposed, but these membrane alterations are largely uncharacterized. In this study, we examined the determinants and characteristics of bile acid-induced membrane alterations, utilizing PKCα activation and cholesterol up-regulation as model indicators. We found that bile acid-induced PKCα activation is a function of hydrophobicity and correlated with alteration in membrane lipid composition, as evident by the significant up-regulation in membrane cholesterol and phospholipid. We found that bile acid do not cause cell membrane disruption at a concentration sufficient to activate PKCα, but do induce drastic alterations in membrane composition. Bile acid also induced the modification and up-regulation of caveolin-1 in a hydrophobicity-dependent manner, implying widespread receptor dysregulation. Similarly, ERK1/2 activation was observed only in response to hydrophobic bile acids, suggesting hydrophobicity-induced caveolar or membrane stress. Experiments with sodium lauryl sarcosine and cholesteryl hemisuccinate showed that bile acid-induced membrane alterations can be mimicked by hydrophobic molecules unrelated to bile acids, strongly implicating hydrophobicity as an important determinant of bile acid signaling.

Keywords: Colon cancer; Bile acid hydrophobicity; Membrane alteration; PKCα; Cholesterol; Caveolin

2n-fatty acids from phosphatidylcholine label sphingolipids—A novel role of phospholipase A₂? by Sybille G.E. Meyer; Werner Karow; Herbert de Groot (pp. 68-78).

In order to find out whether there is a phospholipase A₂ (PLA₂)-mediated link between glycerophospholipids and sphingolipids, L929 cells were labeled with 1n-palmitoyl-2n-[1-¹⁴C]palmitoyl phosphatidylcholine for 16–18 h or 90 min. After labeling for 16–18 h,¹⁴C-sphingomyelin (SM),¹⁴C-ceramide and¹⁴C-sphingosine were demonstrated on autoradiograms of thin layer chromatograms of untreated or mildly hydrolyzed lipid extracts in different chromatographic systems. Strong hydrolysis of labeled SM proved that both possible moieties of SM, sphingosine and acyl moiety, had been labeled. The identity of SM and its enzymatic degradation product, ceramide, was verified by mass spectrometry. The label in SM-derived ceramide was demonstrated on an autoradiogram after thin layer chromatography. The inhibitor of (dihydro)ceramide synthase fumonisin B1 suppressed the label in sphingolipids significantly during 16–18 h (ceramide and SM), as well as during 90-min labeling (SM). The presence of inhibitors of PLA₂ (bromoenol lactone, aristolochic acid and quinacrine dihydrochloride) diminished the label in SM significantly during the 90-min labeling. These results demonstrate a close metabolic relationship between glycerophospholipids and sphingolipids and give evidence for a novel role of PLA₂.

Keywords: Sphingomyelin; Ceramide; Sphingosine; Phosphatidylcholine; Phospholipase A; ₂

Gpi17p does not stably interact with other subunits of glycosylphosphatidylinositol transamidase in Saccharomyces cerevisiae by Yonghua Zhu; Patrick Fraering; Christine Vionnet; Andreas Conzelmann (pp. 79-88).

Homologues of Gpi8p, Gaa1p, Gpi16p, Gpi17p, and Cdc91p are essential components of the GPI transamidase complex that adds glycosylphosphatidylinositols (GPIs 1) to newly synthesized proteins in the ER. In mammalian cells, these five subunits remain stably associated with each other in detergent. In yeast, we find no stable stoichiometric association of Gpi17p with the Gpi8p–Gpi16p–Gaa1p core in detergent extracts. Random and site-directed mutagenesis generated mutations in several highly conserved amino acids but did not yield nonfunctional alleles of Gpi17p and a saturating screen did not yield any dominant negative alleles of Gpi17p. Moreover, Gpi8p becomes unstable when any one of the other subunits is depleted, whereas Gpi17p is slightly affected only by the depletion of Gaa1p. These data suggest that yeast Gpi17p may be able to exert its GPI anchoring function without interacting in a stable and continuous manner with the other GPI-transamidase subunits. Shutting down ER-associated and vacuolar protein degradation pathways has no effect on the levels of Gpi17p or other transamidase subunits.

Keywords: Abbreviations; BCS; bathocuproinedisulfonic acid; GPI; glycosylphosphatidylinositol; NP-40; Nonidet P-40; TMD; transmembrane domain; UPR; unfolded protein response; wt; wild type GPI17; Glycosylphosphatidylinositol; Transamidase; Saccharomyces; Protein stability; Unfolded protein response

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BBA - Molecular and Cell Biology of Lipids (v.1735, #1)