BBA - Molecular and Cell Biology of Lipids (v.1801, #9)

Phosphatidylcholine as a constituent in the colonic mucosal barrier—Physiological and clinical relevance by Robert Ehehalt; Annika Braun; Max Karner; Joachim Füllekrug; Wolfgang Stremmel (983-993).
Phosphatidylcholine (PC) is an important constituent of the gastrointestinal tract. PC molecules are not only important in intestinal cell membranes but also receiving increasing attention as protective agents in the gastrointestinal barrier. They are largely responsible for establishing the hydrophobic surface of the colon. Decreased phospholipids in colonic mucus could be linked to the pathogenesis of ulcerative colitis, a chronic inflammatory bowel disease. Clinical studies revealed that therapeutic addition of PC to the colonic mucus of these patients alleviated the inflammatory activity. This positive role is still elusive, however, we hypothesized that luminal PC has two possible functions: first, it is essential for surface hydrophobicity, and second, it is integrated into the plasma membrane of enterocytes and it modulates the signaling state of the mucosa. The membrane structure and lipid composition of cells is a regulatory component of the inflammatory signaling pathways. In this perspective, we will shortly summarize what is known about the localization and protective properties of PC in the colonic mucosa before turning to its evident medical importance. We will discuss how PC contributes to our understanding of the pathogenesis of ulcerative colitis and how reinforcing the luminal phospholipid monolayer can be used as a therapeutic concept in humans.
Keywords: Phosphatidylcholine; Mucus; Ulcerative colitis; Phospholipids; Mucosal barrier; Lecithin;

The organic solute transporter (OST)(alpha)-OST(beta) is an unusual heteromeric carrier expressed in a variety of tissues including the small intestine, colon, liver, biliary tract, kidney, and adrenal gland. In polarized epithelial cells, OSTα-OSTβ protein is localized on the basolateral membrane and functions in the export or uptake of bile acids and steroids. This article reviews recent results including studies of knockout mouse models that provide new insights to the role of OSTα-OSTβ in the compartmentalization and metabolism of these important lipids.
Keywords: Bile acids; Transporter; Enterocyte; Hepatocyte; Enterohepatic circulation; Malabsorption; Cholestasis; Adrenal; Steroid hormones;

The reduced expression of the bile salt export pump (BSEP/ABCB11) at the canalicular membrane is associated with cholestasis-induced hepatotoxicity due to the accumulation of bile acids in hepatocytes. We previously reported that 4-phenylbutyrate (4PBA), an approved drug for urea cycle disorders, is a promising agent for intrahepatic cholestasis because it increases both the cell surface expression and the transport capacity of BSEP. In the present study, we searched for effective compounds other than 4PBA by focusing on short- and medium-chain fatty acids, which have similar characteristics to 4PBA such as their low-molecular-weight and a carboxyl group. In transcellular transport studies using Madin–Darby canine kidney (MDCK) II cells, all short- and medium-chain fatty acids tested except for formate, acetate, and hexanoic acid showed more potent effects on wild type (WT) BSEP-mediated [3H]taurocholate transport than did 4PBA. The increase in WT BSEP transport with butyrate and octanoic acid treatment correlated with an increase in its expression at the cell surface. Two PFIC2-type variants, E297G and D482G BSEP, were similarly affected with both compounds treatment. The prolonged half-life of cell surface-resident WT BSEP was responsible for this increased octanoic acid-stimulated transport, but not for that of butyrate. In conclusion, short- and medium-chain fatty acids have potent effects on the increase in WT and PFIC2-type BSEP-mediated transport in MDCK II cells. Although both short- and medium-chain fatty acids enhance the transport capacity of WT and PFIC2-type BSEP by inducing those expressions at the cell surface, the underlying mechanism seems to differ between fatty acids.
Keywords: Bile salt export pump (BSEP); Cholestasis; Fatty acid; Bile acid;

Placental ABCA1 and ABCG1 transporters efflux cholesterol and protect trophoblasts from oxysterol induced toxicity by Irving L.M.H. Aye; Brendan J. Waddell; Peter J. Mark; Jeffrey A. Keelan (1013-1024).
ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 mediate the efflux of cholesterol and other sterols. Both transporters are expressed on the fetal capillaries of the placenta and are involved in maternal-to-fetal cholesterol delivery. In this study, we report that ABCA1 and ABCG1 are also present on the syncytiotrophoblast, the maternal facing placental membrane. Syncytial ABCA1 expression is apical, suggesting a role in cholesterol efflux to the mother, while ABCG1 is expressed basolaterally indicating transport to the fetus. Silencing of ABCA1 expression in primary trophoblasts in culture, or pharmacological antagonism by glyburide, decreased cholesterol efflux to apolipoprotein A-I (apoA-I) compared to controls, while ABCG1-silencing decreased cholesterol efflux to high density lipoproteins (HDL). In contrast, treatment with endogenous or synthetic LXR α/β ligands such as T0901317 increased ABCA1 and ABCG1 expression and enhanced cholesterol efflux to apoA-I and HDL, respectively, while treatment with pharmacological PPAR-α or -γ ligands was without effect. Trophoblasts transfected with ABCA1 or ABCG1 siRNA were more sensitive to toxic oxysterols substrates (25-hydroxycholesterol and 7-ketocholesterol) compared to mock-transfected cells, while prior treatment with T0901317 reduced oxysterol-mediated toxicity. These results identify syncytial ABCA1 and ABCG1 as important, inducible cholesterol transporters which also prevent placental accumulation of cytotoxic oxysterols.
Keywords: ABCA1; ABCG1; Cholesterol; LXR; Oxysterol; Placenta;

Lipid-induced up-regulation of human acyl-CoA synthetase 5 promotes hepatocellular apoptosis by Andrea Reinartz; Josef Ehling; Andrea Leue; Christian Liedtke; Ursula Schneider; Jürgen Kopitz; Thomas Weiss; Claus Hellerbrand; Ralf Weiskirchen; Ruth Knüchel; Nikolaus Gassler (1025-1035).
In the pathogenesis of nonalcoholic fatty liver disease, accumulation of lipids in hepatocytes and hepatocyte apoptosis are strongly implicated in disease progression from the potentially reversible condition of steatosis to severe acute and chronic liver injury. Acyl-CoA synthetase 5, a member of the ACSL gene family that catalyzes the activation of long-chain fatty acids for lipid biosynthesis, is the only ACSL isoform that is both, located on mitochondria and functionally involved in enterocyte apoptosis. In this study, the regulation of human ACSL5 in hepatocellular fatty acid degeneration and its involvement in hepatocyte apoptosis was investigated using models of in vitro and in vivo steatosis as well as plasmid-mediated stable gene transfer and RNAi-mediated gene silencing. ACSL5 mRNA and protein were strongly increased by uptake of dietary derived fatty acids in primary human hepatocytes, HepG2 cells and human steatotic liver. Over-expression of ACSL5 decreased HepG2 cell viability and increased susceptibility to TRAIL- and TNFα-, but not FAS- induced apoptosis, whereas knock down of ACSL5 reduced apoptosis susceptibility. High ACSL5 activity resulted in enhanced caspase-3/7 activity, but was not accompanied by up-regulation of death receptors, DR4, DR5 or TNF-R1. This study gives evidence that hepatocyte steatosis is associated with ACSL5 up-regulation resulting in increased susceptibility to hepatic cell death. We propose that ACSL5 could play a role in promoting fatty acid-induced lipoapoptosis in hepatocytes as important mechanism in fatty liver-related disorders.
Keywords: Acyl-CoA synthesis; Lipoapoptosis; Fatty acid metabolism; Steatosis; TNFα; TRAIL;

Lipidomics reveals membrane lipid remodelling and release of potential lipid mediators during early stress responses in a murine melanoma cell line by Gábor Balogh; Mária Péter; Gerhard Liebisch; Ibolya Horváth; Zsolt Török; Enikő Nagy; Andriy Maslyanko; Sándor Benkő; Gerd Schmitz; John L. Harwood; László Vígh (1036-1047).
Membranes are known to respond rapidly to various environmental perturbations by changing their composition and microdomain organization. In previous work we showed that a membrane fluidizer benzyl alcohol (BA) could mimic the effects of heat stress and enhance heat shock protein synthesis in different mammalian cells. Here we explore heat- and BA-induced stress further by characterizing stress-induced membrane lipid changes in mouse melanoma B16 cells. Lipidomic fingerprints revealed that membrane stress achieved either by heat or BA resulted in pronounced and highly specific alterations in lipid metabolism. The loss in polyenes with the concomitant increase in saturated lipid species was shown to be a consequence of the activation of phopholipases (mainly phopholipase A2 and C). A phospholipase C–diacylglycerol lipase–monoacylglycerol lipase pathway was identified in B16 cells and contributed significantly to the production of several lipid mediators upon stress including the potent heat shock modulator, arachidonic acid. The accumulation of cholesterol, ceramide and saturated phosphoglyceride species with raft-forming properties observed upon both heat and BA treatments of B16 cells may explain the condensation of ordered plasma membrane domains previously detected by fluorescence microscopy and may serve as a signalling platform in stress responses or as a primary defence mechanism against the noxious effects of stresses.
Keywords: Lipidomics; Temperature stress; Stress signal; Membrane fluidity; Phospholipase;

Activation of β-adrenergic receptors (AR) in adipocytes triggers acute changes in metabolism that can alter patterns of gene expression. This work examined the mechanisms by which activation of hormone sensitive lipase (HSL) induces expression of inflammatory cytokines in adipocytes in vivo and model adipocytes in vitro. β3-AR activation in mice triggered expression of inflammatory genes CCL2, IL-6, and PAI-1, as well as endoplasmic reticulum (ER) stress markers GRP78 and CHOP. Pharmacological inhibition of HSL blocked induction of inflammatory genes, but not ER stress markers. Promoting intracellular accumulation of free fatty acids (FFAs) in 3T3-L1 adipocytes increased expression of inflammatory cytokines, whereas inhibiting ceramide synthesis partly blocked PAI-1 expression, but not IL-6. Induction of inflammatory markers in vivo and in vitro was preceded by phosphorylation of p38 and JNK, and inhibition of HSL prevented activation of these kinases. Experiments with pharmacological inhibitors of specific MAP kinases demonstrated the importance of p38 MAPK as a mediator of lipolysis-induced inflammation in vivo and in vitro. Together, these results demonstrate that FFAs liberated by HSL activate p38 and JNK, and p38 mediates pro-inflammatory cytokine expression in adipose tissue.
Keywords: Inflammation; β3-adrenergic receptor; Lipolysis; Free fatty acid; Ceramide; Hormone sensitive lipase; P38; JNK;

The current study presents data indicating that 1α,25-dihydroxyvitamin D3 affects the production of hormones and expression of crucial steroidogenic enzymes in the human adrenocortical cell line NCI-H295R. This cell line is widely used as a model for adrenal steroidogenesis. Treatment of the cells with 1α,25-dihydroxyvitamin D3 suppressed the levels of corticosterone, aldosterone, DHEA, DHEA-sulfate and androstenedione in the culture medium. In order to study the mechanisms behind this suppression of hormone production, we investigated the effects of 1α,25-dihydroxyvitamin D3 on important genes and enzymes controlling the biosynthesis of adrenal hormones. The mRNA levels were decreased for CYP21A2 while they were increased for CYP11A1 and CYP17A1. No significant changes were observed in mRNA for CYP11B1, CYP11B2 or 3β-hydroxysteroid dehydrogenase (3βHSD). In similarity with the effects on mRNA levels, also the endogenous enzyme activity of CYP21A2 decreased after treatment with 1α,25-dihydroxyvitamin D3. Interestingly, the two CYP17A1-mediated activities were influenced reciprocally — the 17α-hydroxylase activity increased whereas the 17,20-lyase activity decreased. The current data indicate that the 1α,25-dihydroxyvitamin D3-mediated decrease in corticosterone and androgen production is due to suppression of the 21-hydroxylase activity by CYP21A2 and the 17,20-lyase activity by CYP17A1, respectively. In conclusion, the current study reports novel findings on 1α,25-dihydroxyvitamin D3-mediated effects on hormone production and regulation of genes and enzymes involved in steroidogenesis in the adrenocortical NCI-H295R cell line, a model for human adrenal cortex.
Keywords: Steroidogenesis; Calcitriol; 1α,25-dihydroxyvitamin D3; Regulation;

Identification of Yju3p as functional orthologue of mammalian monoglyceride lipase in the yeast Saccharomyces cerevisiae by Christoph Heier; Ulrike Taschler; Srinivasan Rengachari; Monika Oberer; Heimo Wolinski; Klaus Natter; Sepp D. Kohlwein; Regina Leber; Robert Zimmermann (1063-1071).
Monoacylglycerols (MAGs) are short-lived intermediates of glycerolipid metabolism. Specific molecular species, such as 2-arachidonoylglycerol, which is a potent activator of cannabinoid receptors, may also function as lipid signaling molecules. In mammals, enzymes hydrolyzing MAG to glycerol and fatty acids, resembling the final step in lipolysis, or esterifying MAG to diacylglycerol, are well known; however, despite the high level of conservation of lipolysis, the corresponding activities in yeast have not been characterized yet. Here we provide evidence that the protein Yju3p functions as a potent MAG hydrolase in yeast. Cellular MAG hydrolase activity was decreased by more than 90% in extracts of Yju3p-deficient cells, indicating that Yju3p accounts for the vast majority of this activity in yeast. Loss of this activity was restored by heterologous expression of murine monoglyceride lipase (MGL). Since yju3Δ mutants accumulated MAG in vivo only at very low concentrations, we considered the possibility that MAGs are re-esterified into DAG by acyltransferases. Indeed, cellular MAG levels were further increased in mutant cells lacking Yju3p and Dga1p or Lro1p acyltransferase activities. In conclusion, our studies suggest that catabolic and anabolic reactions affect cellular MAG levels. Yju3p is the functional orthologue of mammalian MGL and is required for efficient degradation of MAG in yeast.
Keywords: Monoacylglycerols; Monoglyceride lipase; MGAT activity; Yeast;

Fish are the primary source in the human food basket of the n-3 long-chain polyunsaturated fatty acids, eicosapentaenoate (EPA; 20:5n-3) and docosahexaenoate (DHA; 22:6n-3), that are crucial to the health of higher vertebrates. Atlantic salmon are able to synthesize EPA and DHA from 18:3n-3 through reactions catalyzed by fatty acyl desaturases (Fad) and elongases of very long chain fatty acids. Previously, two cDNAs encoding functionally distinct Δ5 and Δ6 Fads were isolated, but screening of a genomic DNA library revealed the existence of more putative fad genes in the Atlantic salmon genome. In the present study, we show that there are at least four genes encoding putative Fad proteins in Atlantic salmon. Two genes, Δ6fad_a and Δ5fad, corresponded to the previously cloned Δ6 and Δ5 Fad cDNAs. Functional characterization by heterologous expression in yeast showed that the cDNAs for both the two further putative fad genes, Δ6fad_b and Δ6fad_c, had only Δ6 activity, converting 47 % and 12 % of 18:3n-3 to 18:4n-3, and 25 and 7 % of 18:2n-6 to 18:3n-6, for ∆6Fad_b and Δ6fad_c, respectively. Both ∆6fad_a and ∆6fad_b genes were highly expressed in intestine (pyloric caeca), liver and brain, with ∆6fad_b also highly expressed in gill, whereas ∆6fad_c transcript was found predominantly in brain, with lower expression levels in all other tissues. The expression levels of the ∆6fad_a gene in liver and the ∆6fad_b gene in intestine were significantly higher in fish fed diets containing vegetable oil compared to fish fed fish oil suggesting up-regulation in response to reduced dietary EPA and DHA. In contrast, no significant differences were found between transcript levels for ∆6fad_a in intestine, ∆6fad_b in liver, or ∆6fad_c in liver or intestine of fish fed vegetable oil compared to fish fed fish oil. The observed differences in tissue expression and nutritional regulation of the fad genes are discussed in relation to gene structures and fish physiology.
Keywords: Atlantic salmon; cDNA; Fatty acyl desaturase; Long-chain polyunsaturated fatty acids;

Natural ligand binding and transfer from liver fatty acid binding protein (LFABP) to membranes by Eduardo De Gerónimo; Robert M. Hagan; David C. Wilton; Betina Córsico (1082-1089).
Liver fatty acid-binding protein (LFABP) is distinctive among fatty acid-binding proteins because it binds more than one molecule of long-chain fatty acid and a variety of diverse ligands. Also, the transfer of fluorescent fatty acid analogues to model membranes under physiological ionic strength follows a different mechanism compared to most of the members of this family of intracellular lipid binding proteins. Tryptophan insertion mutants sensitive to ligand binding have allowed us to directly measure the binding affinity, ligand partitioning and transfer to model membranes of natural ligands. Binding of fatty acids shows a cooperative mechanism, while acyl-CoAs binding presents a hyperbolic behavior. Saturated fatty acids seem to have a stronger partition to protein vs. membranes, compared to unsaturated fatty acids. Natural ligand transfer rates are more than 200-fold higher compared to fluorescently-labeled analogues. Interestingly, oleoyl-CoA presents a markedly different transfer behavior compared to the rest of the ligands tested, probably indicating the possibility of specific targeting of ligands to different metabolic fates.
Keywords: Fatty acid; Acyl-CoA; Lipid binding protein; Lipid transfer; Enterocyte; Lipid metabolism;

Effects of CYP7B1-mediated catalysis on estrogen receptor activation by Hanna Pettersson; Johan Lundqvist; Maria Norlin (1090-1097).
Most of the many biological effects of estrogens are mediated via the estrogen receptors ERα and β. The current study examines the role of CYP7B1-mediated catalysis for activation of ER. Several reports suggest that CYP7B1 may be important for hormonal action but previously published studies are contradictory concerning the manner in which CYP7B1 affects ERβ-mediated response. In the current study, we examined effects of several CYP7B1-related steroids on ER activation, using an estrogen response element (ERE) reporter system. Our studies showed significant stimulation of ER by 5-androstene-3β,17β-diol (Aene-diol) and 5α-androstane-3β,17β-diol (3β-Adiol). In contrast, the CYP7B1-formed metabolites from these steroids did not activate the receptor, indicating that CYP7B1-mediated metabolism abolishes the ER-stimulating effect of these compounds. The mRNA level of HEM45, a gene known to be stimulated by estrogens, was strongly up-regulated by Aene-diol but not by its CYP7B1-formed metabolite, further supporting this concept. We did not observe stimulation by dehydroepiandrosterone (DHEA) or 7α-hydroxy-DHEA, previously suggested to affect ERβ-mediated response. As part of these studies we examined metabolism of Aene-diol in pig liver which is high in CYP7B1 content. These experiments indicate that CYP7B1-mediated metabolism of Aene-diol is of a similar rate as the metabolism of the well-known CYP7B1 substrates DHEA and 3β-Adiol. CYP7B1-mediated metabolism of 3β-Adiol has been proposed to influence ERβ-mediated growth suppression. Our results indicate that Aene-diol also might be important for ER-related pathways. Our data indicate that low concentrations of Aene-diol can trigger ER-mediated response equally well for both ERα and β and that CYP7B1-mediated conversion of Aene-diol into a 7α-hydroxymetabolite will result in loss of action.
Keywords: Estrogen; CYP7B1; Hydroxylation; ER-mediated response; Steroid metabolism;

Endogenous H2O2 produced by Streptococcus pneumoniae controls FabF activity by Rachel Benisty; Aharon Yehonatan Cohen; Alexandra Feldman; Zvi Cohen; Nurith Porat (1098-1104).
FabF elongation condensing enzyme is a critical factor in determining the spectrum of products produced by the FASII pathway. Its active site contains a critical cysteine-thiol residue, which is a plausible target for oxidation by H2O2. Streptococcus pneumoniae produces exceptionally high levels of H2O2, mainly through the conversion of pyruvate to acetyl-P via pyruvate oxidase (SpxB). We present evidence showing that endogenous H2O2 inhibits FabF activity by specifically oxidizing its active site cysteine-thiol residue. Thiol trapping methods revealed that one of the three FabF cysteines in the wild-type strain was oxidized, whereas in an spxB mutant, defective in H2O2 production, none of the cysteines was oxidized, indicating that the difference in FabF redox state originated from endogenous H2O2. In vitro exposure of the spxB mutant to various H2O2 concentrations further confirmed that only one cysteine residue was susceptible to oxidation. By blocking FabF active site cysteine with cerulenin we show that the oxidized cysteine was the catalytic one. Inhibition of FabF activity by either H2O2 or cerulenin resulted in altered membrane fatty acid composition. We conclude that FabF activity is inhibited by H2O2 produced by S. pneumoniae.►Endogenous H2O2 produced by Streptococcus pneumoniae inhibits FabF activity by oxidizing its active site cysteine-thiol residue. ►Inhibition of FabF activity results in a significant change in membrane fatty acid composition, mainly: decreased proportions of long, unsaturated fatty acyl chains. ►The decrease in the proportions of long, unsaturated fatty acyl chains induces the transcription of FASII genes. ►The present study provides evidence showing that H2O2 produced by S. pneumoniae serves as a beneficial molecular signal, allowing the attenuation of FabF activity, to enable membrane adaptation at various growth conditions.
Keywords: FabF; FASII; Redox state; Cerulenin; Streptococcus pneumoniae; Membrane fatty acid;

Corrigendum to “Gelucire® 44/14 improves fat absorption in rats with impaired lipolysis” [Biochim. Biophys. Acta 1801 (2010) 665–673] by S. Lukovac; K.E.R. Gooijert; P.C. Gregory; G. Shlieout; F. Stellaard; E.H.H.M. Rings; H.J. Verkade (1105).