BBA - Molecular and Cell Biology of Lipids (v.1811, #12)

Tight association of N-terminal and catalytic subunits of rabbit 12/15-lipoxygenase is important for protein stability and catalytic activity by Igor Ivanov; Almerinda Di Venere; Thomas Horn; Patrick Scheerer; Eleonora Nicolai; Sabine Stehling; Constanze Richter; Ewa Skrzypczak-Jankun; Giampiero Mei; Mauro Maccarrone; Hartmut Kühn (1001-1010).
12/15-Lipoxygenases (12/15-LOXs) have been implicated in inflammatory and hyperproliferative diseases but the structural biology of these enzymes is not well developed. Most LOXs constitute single polypeptide chain proteins that fold into a two-domain structure. In the crystal structure the two domains are tightly associated, but small angle X-ray scattering data and dynamic fluorescence studies suggested a high degree of structural flexibility involving movement of the N-terminal domain relative to catalytic subunit. When we inspected the interdomain interface we have found a limited number of side-chain contacts which are involved in interactions of these two structural subunits. One of such contact points involves tyrosine 98 of N-terminal domain. This aromatic amino acid is invariant in vertebrate LOXs regardless of overall sequence identity. To explore in more detail the role of aromatic interactions in interdomain association we have mutated Y98 to various residues and quantified the structural and functional consequences of these alterations. We have found that loss of an aromatic moiety at position 98 impaired the catalytic activity and membrane binding capacity of the mutant enzymes. Although CD and fluorescence emission spectra of wild-type and mutant enzyme species were indistinguishable, the mutation led to enlargement of the molecular shape of the enzyme as detected by analytic gel filtration and this structural alteration was shown to be associated with a loss of protein thermal stability. The possible role of tight interdomain association for the enzyme's structural performance is discussed.Display Omitted► Tight interdomain association is important for structural performance of 12/15-LOX. ► Tyr98 is involved in aromatic interaction between two subunits of 12/15-LOX. ► A loss of an aromatic interaction destabilize tertiary structure of 12/15-LOX. ► This structural alteration is associated with a functional distortion of 12/15-LOX.
Keywords: Eicosanoids; Lipid peroxidation; Molecular enzymology; Structural biology; Motional flexibility; Thermal stability;

Lack of acyl-CoA:diacylglycerol acyltransferase 1 reduces intestinal cholesterol absorption and attenuates atherosclerosis in apolipoprotein E knockout mice by Prakash G. Chandak; Sascha Obrowsky; Branislav Radovic; Prakash Doddapattar; Elma Aflaki; Adelheid Kratzer; Lalit S. Doshi; Silvia Povoden; Helmut Ahammer; Gerald Hoefler; Sanja Levak-Frank; Dagmar Kratky (1011-1020).
Triacylglycerols (TG) are the major storage molecules of metabolic energy and fatty acids in several tissues. The final step in TG biosynthesis is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. Lack of whole body DGAT1 is associated with reduced lipid-induced inflammation. Since one major component of atherosclerosis is chronic inflammation we hypothesized that DGAT1 deficiency might ameliorate atherosclerotic lesion development. We therefore crossbred Apolipoprotein E-deficient (ApoE −/− ) mice with Dgat1 −/− mice. ApoE −/− and ApoE −/− Dgat1 −/− mice were fed Western-type diet (WTD) for 9 weeks and thereafter examined for plaque formation. The mean atherosclerotic lesion area was substantially reduced in ApoE −/− Dgat1 −/− compared with ApoE −/− mice in en face and aortic valve section analyses. The reduced lesion size was associated with decreased cholesterol uptake and absorption by the intestine, reduced plasma TG and cholesterol concentrations and increased cholesterol efflux from macrophages. The expression of adhesion molecules was reduced in aortas of ApoE −/− Dgat1 −/− mice, which might be the reason for less migration capacities of monocytes and macrophages and the observed decreased amount of macrophages within the plaques. From our results we conclude that the lack of DGAT1 is atheroprotective, implicating an additional application of DGAT1 inhibitors with regard to maintaining cholesterol homeostasis and attenuating atherosclerosis.Display Omitted► DGAT1 deficiency on an ApoE-null background reduces diet-induced atherosclerotic lesion formation. ► DGAT1 deficiency decreases cholesterol uptake and absorption in ApoE-null mice. ► Reduced macrophage migration and decreased aortic inflammation in ApoE-null mice lacking DGAT1. ► Increased cholesterol efflux from macrophages lacking DGAT1. ► Additional potential application of DGAT1 inhibitors with regard to attenuating atherosclerosis.
Keywords: Atherosclerosis; Cholesterol absorption; Cholesterol efflux; Acyl-CoA:diacylglycerol acyltransferase 1; Inflammation; Apolipoprotein E knockout mice;

Sphingosylphosphorylcholine (SPC) is found at increased levels in the malignant ascites of tumor patients and induces perinuclear reorganization of keratin 8 (K8) filaments that contribute to the viscoelasticity of metastatic cancer cells. In this study, we investigated the role and molecular mechanisms of Tgase-2 in SPC-induced K8 phosphorylation and perinuclear reorganization in PANC-1 cells (PANWT), and in PANC-1 cells that stably expressed shTgase-2 or Tgase-2 (PANshTg2 and PANTg2). SPC induces the expression of Tgase-2 in a time- and dose-dependent manner. Gene silencing of Tgase-2 or cystamine suppressed the SPC-induced phosphorylation and perinuclear reorganization of K8 and suppressed the SPC-induced migration of PANC-1 cells. An inhibitor of c-Jun N-terminal kinase (JNK), SP600125, suppressed the SPC-induced phosphorylation of serine 431 in K8 and keratin reorganization. Next, we examined the effect of Tgase-2 on JNK activation of serine 431 phosphorylation in K8. Tgase-2 gene silencing suppressed the expression of active form JNK (pJNK). Constitutive or tetracyclin-induced conditional expression of Tgase-2 increased the levels of pJNK. Tgase-2 was coimmunoprecipitated with K8 and JNK. In addition, K8 was coimmunoprecipitated with Tgase-2 and JNK. JNK was also coimmunoprecipitated with K8 and Tgase-2. Overall, these results suggest that Tgase-2 is involved in SPC-induced phosphorylation and perinuclear reorganization of K8 by activating JNK and forming a triple complex with K8 and JNK. Therefore, SPC-induced Tgase-2 might be a new target for modulating keratin reorganization, metastasis of cancer cells and JNK activation.Display Omitted► SPC induces expression of Tgase-2 in time and dose-dependent manner. ► Tgase-2 is involved in SPC-induced Ser 431 phosphorylation of keratin-8. ► JNK activation is dependent on Tgase-2 in SPC-induced Ser 431 phosphorylation.
Keywords: Sphingosylphosphorylcholine; Transglutaminase-2; Keratin-8 phosphorylation and reorganization; c-Jun N-terminal kinase; Migration;

Metabolic link between phosphatidylethanolamine and triacylglycerol metabolism in the yeast Saccharomyces cerevisiae by Susanne E. Horvath; Andrea Wagner; Ernst Steyrer; Günther Daum (1030-1037).
In the yeast Saccharomyces cerevisiae triacylglycerols (TAG) are synthesized by the acyl-CoA dependent acyltransferases Dga1p, Are1p, Are2p and the acyl-CoA independent phospholipid:diacylglycerol acyltransferase (PDAT) Lro1p which uses phosphatidylethanolamine (PE) as a preferred acyl donor. In the present study we investigated a possible link between TAG and PE metabolism by analyzing the contribution of the four different PE biosynthetic pathways to TAG formation, namely de novo PE synthesis via Psd1p and Psd2p, the CDP-ethanolamine (CDP-Etn) pathway and lyso-PE acylation by Ale1p. In cells grown on the non-fermentable carbon source lactate supplemented with 5 mM ethanolamine (Etn) the CDP-Etn pathway contributed most to the cellular TAG level, whereas mutations in the other pathways displayed only minor effects. In cki1∆dpl1∆eki1∆ mutants bearing defects in the CDP-Etn pathway both the cellular and the microsomal levels of PE were markedly decreased, whereas in other mutants of PE biosynthetic routes depletion of this aminoglycerophospholipid was less pronounced in microsomes. This observation is important because Lro1p similar to the enzymes of the CDP-Etn pathway is a component of the ER. We conclude from these results that in cki1∆dpl1∆eki1∆ insufficient supply of PE to the PDAT Lro1p was a major reason for the strongly reduced TAG level. Moreover, we found that Lro1p activity was markedly decreased in cki1∆dpl1∆eki1∆, although transcription of LRO1 was not affected. Our findings imply that (i) TAG and PE syntheses in the yeast are tightly linked; and (ii) TAG formation by the PDAT Lro1p strongly depends on PE synthesis through the CDP-Etn pathway. Moreover, it is very likely that local availability of PE in microsomes is crucial for TAG synthesis through the Lro1p reaction.► CDP-Etn pathway provides PE for Lro1p. ► Depletion of PE in ER causes low TAG. ► Link between phospholipid and TAG metabolism.
Keywords: Phosphatidylethanolamine; Triacylglycerol; Acyltransferase; CDP-ethanolamine; Yeast; Saccharomyces cerevisiae;

Cholesterol and membrane phospholipid compositions modulate the leakage capacity of the swaposin domain from a potato aspartic protease (StAsp-PSI) by Fernando Muñoz; M. Francisca Palomares-Jerez; Gustavo Daleo; José Villalaín; M. Gabriela Guevara (1038-1044).
Potato aspartic proteases (StAPs) and their swaposin domain (StAsp-PSI) are proteins with cytotoxic activity which involves plasma membrane destabilization. The ability of these proteins to produce cell death varies with the cellular type. Therefore, StAPs and StAsp-PSI selective cytotoxicity could be attributed to the different membrane lipid compositions of target cells. In this work we investigate the possible mechanism by which StAPs and StAsp-PSI produce selective membrane destabilization. Results obtained from leakage assays show that StAsp-PSI is a potent inducer of the leakage of LUVs containing anionic phospholipids, especially those containing phosphatidylglycerol. Based in these results, we suggest that the cytotoxic activity of StAsp-PSI on pathogenic microorganisms could be mediated by the attraction between the exposed positive domains of StAsp-PSI and the negatively charged microorganism membrane. On the other hand, our circular dichroism spectroscopic measurements and analysis by size exclusion chromatography and followed by electrophoresis, indicate that hydrophobic environment is necessary to StAsp-PSI oligomerization and both StAsp-PSI disulfide bounds and membrane with negative charged phospholipids are required by StAsp-PSI to produce membrane destabilization and then induce cell death in tumors and microorganism cell targets. Additionally, we demonstrate that the presence of cholesterol into the LUV membranes strongly diminishes the capacity of StAsp-PSI to produce leakage. This result suggests that the lack of hemolytic and cytotoxic activities on human lymphocytes of StAsp-PSI/StAPs may be partly due by the presence of cholesterol in these cell membrane types. ► StAsp-PSI cytotoxicity requires disulfide bounds and negative charge phospholipids. ► Oligomerization of StAsp-PSI is necessary for its cytotoxic activity. ► StAsp-PSI cytotoxicity is inhibited by cholesterol.
Keywords: Antimicrobial protein; Antitumor protein; Plant aspartic protease; Membrane destabilization; α-helix protein; Plat defense response;

Conflicting data have been reported regarding the role of retinol-binding protein (RBP4) in insulin resistance, obesity, type 2 diabetes and non-alcoholic fatty liver disease (NAFLD). In this study, we used pharmacological methods to investigate the role of RBP4. RNA oligonucleotide against RBP4 (anti-RBP4 oligo) was transfected into 3T3-L1 adipocytes. RT-PCR analysis showed that RBP4 mRNA expression decreased by 55% (p  < 0.01) compared with control cells. Validated RNA oligo was used in an in vivo study with high fat diet (HFD) fed — mice. 14 weeks of HFD feeding increased RBP4 expression (associated with elevated serum levels measured with immunoblotting and ELISA) by 56% in adipose tissue (p  < 0.05) and 68% in the liver (p  < 0.01). Adipose RBP4 levels were significantly reduced after 4 weeks treatment with anti-RBP4 oligo (25 mg/kg, p  < 0.01) and rosiglitazone (RSG, 10 mg/kg, p  < 0.05) compared with scrambled RNA oligo (25 mg/kg) treated mice. Only anti-RBP4 oligo significantly inhibited RBP4 protein (p  < 0.01) and mRNA expression (p  < 0.01) in the liver and reduced serum RBP4 levels. Anti-RBP4 oligo and RSG showed comparable effects on impaired glucose tolerance, hyperinsulinaemia and hyperglycaemia. Anti-RBP4 oligo significantly enhanced adipose-GLUT4 expression (p  < 0.01) but did not increase muscle-GLUT4. Both RSG and anti-RBP4 oligo significantly reduced hepatic phosphoenolpyruvate carboxykinase expression (both p  < 0.05). Histological analysis revealed that anti-RBP4 oligo ameliorated hepatic steatosis and reduced lipid droplets associated with normalized liver function. Histological and pharmacological results of this study indicate that RBP4 is not only an adipocytokine, but also a hepatic cytokine leading to metabolic syndrome, NAFLD and type 2 diabetes.► Over-expression of RBP4 leads to insulin resistance and fatty liver in HFD mice. ► RNA oligonucleotide targeted at RBP4 ameliorates metabolic syndrome in HFD mice. ► Anti-RBP4 RNA oligonucleotide reduces liver fat and improves hepatic steatosis.
Keywords: Retinol-binding protein 4; Anti-RBP4 RNA oligonucleotide; RBP4 inhibition; High fat diet fed mice; Metabolic syndrome; Non-alcoholic fatty liver disease;

Corynespora cassiicola has a unique but unexplored ability amongst fungi, in that it can hydroxylate 17α-hydroxyprogesterone at the highly hindered C-8 position of the steroid nucleus. In order to gain greater understanding of the mechanistic basis and capability of the 8β-hydroxylase we have transformed a range of structurally diverse androgens and progestogens with this organism. This has revealed that both steroid types can be hydroxylated at the 8β-position. The collective data has demonstrated the first time that 8β-hydroxylation occurs through inverted binding within a 9α-hydroxylase of the fungus. In the case of the progestogens, for this to occur, the presence of 17α-oxygen functionality (alcohol or epoxide) was essential. Remarkably monohydroxylation of 17α-hydroxyprogesterone at carbons 8β and 15β has strongly indicated that the responsible hydroxylase has 2 different binding sites for the ring-A ketone. Unusually, with one exception, all hydroxylation occurred at axial protons and in the case of the progestogens, all above the plane of the ring system. In general all maximally oxidised metabolites contained four oxygen atoms. The importance of these findings in relation to 8β-hydroxylation of these steroids is discussed.► 8β-Hydroxylation occurs through capsized binding in a 9α-hydroxylase. ► 17α-Oxygen functionality required for 8β-hydroxylation of progestogens. ► First single crystal X-ray structural determination of 8β,17α-dihydroxyprogesterone. ► 17α-Hydroxyprogesterone hydroxylation indicates C-3 ketone has two binding sites.
Keywords: Androgen; Biocatalyst; Corynespora cassiicola; 8β-Hydroxylation; Dual binding region; Progestogen;

ApoA-I enhances generation of HDL-like lipoproteins through interaction between ABCA1 and phospholipase Cγ in rat astrocytes by Jin-ichi Ito; Yuko Nagayasu; Alireza Kheirollah; Sumiko Abe-Dohmae; Shinji Yokoyama (1062-1069).
In the previous paper, we reported that apolipoprotein (apo) A-I enhances generation of HDL-like lipoproteins in rat astrocytes to be accompanied with both increase in tyrosine phosphorylation of phospholipase Cγ (PL-Cγ) and PL-Cγ translocation to cytosolic lipid–protein particles (CLPP) fraction. In this paper, we studied the interaction between apoA-I and ATP-binding cassette transporter A1 (ABCA1) to relate with PL-Cγ function for generation of HDL-like lipoproteins in the apoA-I-stimulated astrocytes. ABCA1 co-migrated with exogenous apoA-I with apparent molecular weight over 260 kDa on SDS-PAGE when rat astrocytes were treated with apoA-I and then with a cross-linker, BS3. The solubilized ABCA1 of rat astrocytes was associated with the apoA-I-immobilized Affi-Gel 15. An LXR agonist, To901317, increased the cellular level of ABCA1, association of apoA-I with ABCA1 and apoA-I-mediated lipid release in rat astrocytoma GA-1/Mock cells where ABCA1 expression at baseline is very low. PL-Cγ was co-isolated by apoA-I-immobilized Affi-Gel 15 and co-immunoprecipitated by anti-ABCA1 antibody along with ABCA1 from the solubilized membrane fraction of rat astrocytes. The SiRNA of ABCA1 suppressed not only the PL-Cγ binding to ABCA1 but also the tyrosine phosphorylation of PL-Cγ. A PL-C inhibitor, U73122, prevented generation of apoA-I-mediated HDL-like lipoproteins in rat astrocytes. To901317 increased the association of PL-Cγ with ABCA1 in GA-1/Mock cells dependently on the increase of cellular level of ABCA1 without changing that of PL-Cγ. These findings suggest that the exogenous apoA-I augments the interaction between PL-Cγ and ABCA1 to stimulate tyrosine phosphorylation and activation of PL-Cγ for generation of HDL-like lipoproteins in astrocytes.► We investigate an apoA-I receptor in the CNS and the mechanism of action. ► We show that ABCA1 is an apoA-I-binding protein of astrocytes. ► ABCA1 enhances the association of CLPP and microtubules through the interaction with PL-Cγ.
Keywords: ApoA-I; Astrocyte; ABCA1; Phospholipase Cγ; HDL;

Metabolic and growth inhibitory effects of conjugated fatty acids in the cell line HT-29 with special regard to the conversion of t11,t13-CLA by Christian Degen; Josef Ecker; Stefanie Piegholdt; Gerhard Liebisch; Gerd Schmitz; Gerhard Jahreis (1070-1080).
Conjugated fatty acids (CFAs) exhibit growth inhibitory effects on colon cancer in vitro and in vivo. To investigate whether the anticancerogenic potency depends on number or configuration of the conjugated double bonds, the effect of conjugated linoleic acid (CLA; C18:2) isomers and conjugated linolenic acid (CLnA; C18:3) isomers on viability and growth of HT-29 cells were compared. Low concentrations of CLnAs (< 10 μM) yielded a higher degree of inhibitory effects compared to CLAs (40 μM). All trans-CFAs were more effective compared to cis/trans-CFAs as follows: t9,t11,t13-CLnA ≥  c9,t11,t13-CLnA >  t11,t13-CLA ≥  t9,t11-CLA >  c9,t11-CLA. The mRNA expression analysis of important genes associated with fatty acid metabolism showed an absence of ∆5-/∆6-desaturases and elongases in HT-29 cells, which was confirmed by fatty acid analysis. Using time- and dose-dependent stimulation experiments several metabolites were determined. Low concentrations of all trans-CFAs (5–20 μM) led to dose-dependent increase of conjugated t/t-C16:2 formed by β-oxidation of C18 CFAs, ranging from 1–5% of total FAME. Importantly, it was found that CLnA is converted to CLA and that CLA is inter-converted (t11,t13-CLA is metabolized to c9,t11-CLA) by HT-29 cells. In summary, our study shows that growth inhibition of human cancer cells is associated with a specific cellular transcriptomic and metabolic profile of fatty acid metabolism, which might contribute to the diversified ability of CFAs as anti-cancer compounds.► CLnA inhibited viability and cell growth of HT-29 more effective than CLA. ► All trans-CFAs were more effective compared to cis/trans-CFAs. ► Investigation of genes revealed absence of ∆5-/∆6-desaturases and elongases. ► This study confirmed conversion of CLnA isomers to CLA isomers. ► The conversion of CLA (t11,t13) into another CLA isomer (c9,t11) was shown.
Keywords: Conjugated linoleic acid; Conjugated linolenic acid; Isomer specificity; Conversion; Colon cancer cell;

Quantitative profiling of PE, MMPE, DMPE, and PC lipid species by multiple precursor ion scanning: A tool for monitoring PE metabolism by Mesut Bilgin; Daniel F. Markgraf; Eva Duchoslav; Jens Knudsen; Ole N. Jensen; Anton I.P.M. de Kroon; Christer S. Ejsing (1081-1089).
We report a method for the simultaneous identification and quantification of phosphatidylethanolamine (PE), monomethyl-phosphatidylethanolamine (MMPE), dimethyl-phosphatidylethanolamine (DMPE), and phosphatidylcholine (PC) species in lipid extracts. The method employs a specific “mass-tag” strategy where DMPE, MMPE, and PE species are chemically methylated with deuterated methyliodide (CD3I) to produce PC molecules having class-specific mass offsets of 3, 6 and 9 Da, respectively. The derivatized aminoglycerophospholipids release characteristic phosphorylcholine-like fragment ions having specific mass offsets that powers sensitive and quantitative analysis by multiple precursor ion scanning on a hybrid quadrupole time-of-flight mass spectrometer. Using the mass-tag strategy, we could for the first time determine the stoichiometric relationship between the biosynthetic intermediates MMPE and DMPE, and abundant PE and PC species in a single mass spectrometric analysis. We demonstrated the efficacy of the methodology by conducting a series of biochemical experiments using stable isotope labeled ethanolamine to survey the activities and substrate specificities of enzymes involved in PE metabolism in Saccharomyces cerevisiae. Finally, we benchmarked the mass-tag strategy by specific and sensitive profiling of intermediate MMPE and DMPE species in liver.► We present a new lipidomic routine for PE, MMPE, DMPE, and PC lipid species. ► A mass-tag strategy is applied to convert PE, MMPE, and DMPE into PC-like molecules. ► Simultaneous, specific and sensitive analysis of PE, MMPE, DMPE, and PC species. ► The methodology affords dynamic lipidomic studies of PE metabolism.
Keywords: Shotgun lipidomics; Chemical derivatization; Aminoglycerophospholipids; Quadrupole time-of-flight mass spectrometry; Multiple precursor ion scanning; Saccharomyces cerevisiae;

Identification and characterization of ABCB1-mediated and non-apoptotic sebum secretion in differentiated hamster sebocytes by Hirokazu Kurihara; Takashi Sato; Noriko Akimoto; Takayuki Ogura; Akira Ito (1090-1096).
Sebaceous glands secrete sebum onto the skin surface in a holocrine manner and as such a thin lipid layer is formed as a physiological barrier. In the present study, extracellular level of triacylglycerols (TG), a major sebum component, as well as intracellular TG accumulation was augmented in insulin-differentiated hamster sebocytes (DHS). The DHS exhibited phosphatidylserine exposure in an apoptosis-independent manner. In addition, intracellular ATP level and membrane-transporter activity using a substrate, Rhodamine 123, were highly detectable in the DHS rather than in the undifferentiated hamster sebocytes. A membrane-transporter activating reagent, 2′(3′)-O-(4-benzoylbenzoyl) adenosine 5′-triphosphate (BzATP), enhanced transporter activity, extracellular TG level, and phosphatidylserine exposure in the DHS. Both transporter activity and TG secretion were suppressed by R-verapamil, a potent membrane-transporter inhibitor, in the BzATP-treated and untreated DHS. Furthermore, the gene expression and production of ATP-binding cassette subfamily B member 1 (ABCB1) were augmented in the DHS. ABCB1 was also detectable in sebaceous glands in the skin of hamsters. Moreover, the cell-differentiation- and BzATP-augmented transporter activity and TG secretion were dose-dependently inhibited by adding not only an ABCB1 antibody but also a selective inhibitor of ABCB1, PSC833. Thus, these results provide novel evidence that ABCB1 is involved in sebum secretion in the DHS, which is associated with non-apoptotic phosphatidylserine exposure and the increased level of intracellular ATP. These findings should accelerate the understanding of sebum secretion occurring in a holocrine-independent manner in sebaceous glands, and may contribute to the development of therapies for sebaceous gland disorders such as acne, seborrhea, and xerosis.►Sebocyte differentiation results in the augmentation of sebum secretion. ►Differentiated sebocytes exhibit apoptosis-independent PS exposure. ►Increase in ATP level and transporter activity in differentiated sebocytes. ►ABCB1 expression is augmented in differentiated sebocytes. ►Involvement of ABCB1 in sebum secretion in differentiated sebocytes.
Keywords: Sebum secretion; Sebocyte; Phosphatidylserine exposure; ABC transporter; ABCB1; ATP;

Complex modulation of peptidolytic activity of cathepsin D by sphingolipids by Iva Žebrakovská; Martin Máša; Jaroslav Srp; Martin Horn; Kateřina Vávrová; Michael Mareš (1097-1104).
Cathepsin D is an aspartic peptidase involved in cellular processes including proliferation and apoptosis and implicated in human pathologies such as cancer and neurodegeneration. Our knowledge about the relationship between proteolysis and bioactive sphingolipids is still very limited. Here, we describe a complex pattern of modulation of the peptidolytic activity of cathepsin D by sphingolipids. A panel of sphingolipid derivatives was screened in a FRET-based assay; these molecules demonstrated negative or positive modulation of cathepsin D peptidolytic activity, depending on the sphingolipid structure. Certain sphingosines and ceramides inhibited cathepsin D in the submicromolar range, and structural requirements for this inhibitory effect were evaluated. The interaction of cathepsin D with sphingolipids was also demonstrated by fluorescence polarization measurements and determined to follow a competitive inhibition mode. In contrast, monoester phosphosphingolipids, especially ceramide-1-phosphate, were identified as activators of cathepsin D peptidolytic activity at submicromolar concentrations. Thus, sphingolipids and phosphosphingolipids, known to be antagonistic in their cell-signaling functions, displayed opposite modulation of cathepsin D. Sphingolipid-based modulators of cathepsin D are potentially involved in the control of cathepsin D-dependent processes and might serve as a scaffold for the development of novel regulators of this therapeutic target.► Sphingolipids are complex modulators of peptidolytic activity of cathepsin D. ► This modulatory interaction is potentially relevant to apoptosis and proliferation. ► Sphingosines and ceramides are the first endogenous inhibitors of cathepsin D. ► Monoester phosphosphingolipids activate cathepsin D.
Keywords: Sphingolipid; Phospholipid; Inhibition; Activation; Cathepsin D; Enzyme regulation;

Apo-10'-lycopenoic acid impacts adipose tissue biology via the retinoic acid receptors by E. Gouranton; G. Aydemir; E. Reynaud; J. Marcotorchino; C. Malezet; C. Caris-Veyrat; R. Blomhoff; J.F. Landrier; R. Rühl (1105-1114).
Apo-10'-lycopenoic acid (apo-10-lycac), a metabolite of lycopene, has been shown to possess potent biological activities, notably via the retinoic acid receptors (RAR). In the current study, its impact on adipose tissue and adipocytes was studied. In microarray experiments, the set of genes regulated by apo-10-lycac treatments was compared to the set of genes regulated by all-trans retinoic acid (ATRA), the natural ligand of RAR, in adipocytes. Approximately 27.5% of the genes regulated by apo-10-lycac treatments were also regulated by ATRA, suggesting a common ability in terms of gene expression modulation, possibly via RAR transactivation. The physiological impact of apo-10-lycac on adipose tissue biology was evaluated. If it had no effect on adipogenesis in the 3T3-L1 cell model, this metabolite may have a preventative effect against inflammation, by preventing the increase in the inflammatory markers, interleukin 6 and interleukin 1β in various dedicated models. The ability of apo-10-lycac to transactivate the RAR and to modulate the transcription of RAR target gene was brought in vivo in adipose tissue. While apo-10-lycac was not detected in adipose tissue, a metabolite with a molecular weight with 2 Da larger mass was detected, suggesting that a dihydro-apo-10'-lycopenoic acid, may be present in adipose tissue and that this compound could active or may lead to further active RAR-activating apo-10-lycac metabolites. Since apo-10-lycac treatments induce anti-inflammatory effects in adipose tissue but do not inhibit adipogenesis, we propose that apo-10-lycac treatments and its potential active metabolites in WAT may be considered for prevention strategies relevant for obesity-associated pathologies.► Apo-10'-lycopenoic acid displays similarities in term of gene expression with all-trans retinoic acid. ► Apo-10'-lycopenoid acid reduces TNFα and high fat mediated inflammatory response in adipocytes and adipose tissue. ► Apo-10'-lycopenoic acid transactivates RAR in adipose tissue of reporter mice. ► Apo-10'-lycopenoic acid modulates the expression of RAR target genes in vivo.
Keywords: Lycopene; apo-10'-lycopenoic acid; Adipocytes; Adipose tissue; Inflammation; RAR;

Trimerized apolipoprotein A-I (TripA) forms lipoproteins, activates lecithin:cholesterol acyltransferase, elicits lipid efflux, and is transported through aortic endothelial cells by Pascale M. Ohnsorg; Jean-Luc Mary; Lucia Rohrer; Michael Pech; Jürgen Fingerle; Arnold von Eckardstein (1115-1123).
Apolipoprotein A-I (apoA-I) exerts many potentially anti-atherogenic properties and is therefore attractive for prevention and therapy of coronary heart disease. Since induction of apoA-I production by small molecules has turned out as difficult, application of exogenous apoA-I is pursued as an alternative therapeutic option. To counteract fast renal filtration of apoA-I, a trimeric high-molecular weight variant of apoA-I (TripA) was produced by recombinant technology. We compared TripA and apoA-I for important properties in reverse cholesterol transport. Reconstituted high-density lipoproteins (rHDL) containing TripA or apoA-I together with palmitoyl-2-oleyl-phosphatidylcholine (POPC) differed slightly by size. Compared to apoA-I, TripA activated lecithin:cholesterol acyltransferase (LCAT) with similar maximal velocity but concentration leading to half maximal velocity was slightly reduced (Km  = 2.1 ± 0.3 μg/mL vs. 0.59 ± 0.06 μg/mL). Both in the lipid-free form and as part of rHDL, TripA elicited cholesterol efflux from THP1-derived macrophages with similar kinetic parameters and response to liver-X-receptor activation as apoA-I. Lipid-free TripA is bound and transported by aortic endothelial cells through mechanisms which are competed by apoA-I and TripA and inhibited by knock-down of ATP-binding cassette transporter (ABC) A1. Pre-formed TripA/POPC particles were bound and transported by endothelial cells through mechanisms which are competed by excess native HDL as well as reconstituted HDL containing either apoA-I or TripA and which involve ABCG1 and scavenger receptor B1 (SR-BI). In conclusion, apoA-I and TripA show similar in vitro properties which are important for reverse cholesterol transport. These findings are important for further development of TripA as an anti-atherosclerotic drug.► Apolipoprotein A-I (apoA-I) and HDL exert potentially anti-atherogenic properties. ► Trimerized apoA-(TripA) is a potentially therapeutic mimetic of apoA-I and HDL. ► TripA resembles apoA-I by LCAT and cholesterol efflux stimulating properties. ► TripA resembles apoA-I by binding and transport through endothelial cells.
Keywords: High-density lipoprotein; Endothelium; Transcytosis; Reverse cholesterol transport; Cholesterol efflux; ABCA1;

Brain Specific Kinase-1 BRSK1/SAD-B associates with lipid rafts: modulation of kinase activity by lipid environment by Arantza Rodríguez-Asiain; Gerard Ruiz-Babot; Walter Romero; Roger Cubí; Tatiana Erazo; Ricardo M. Biondi; Jose R. Bayascas; Jose Aguilera; Nestor Gómez; Carles Gil; Enrique Claro; Jose M. Lizcano (1124-1135).
Brain specific kinases 1 and 2 (BRSK1/2, also named SAD kinases) are serine–threonine kinases specifically expressed in the brain, and activated by LKB1-mediated phosphorylation of a threonine residue at their T-loop (Thr189/174 in human BRSK1/2). BRSKs are crucial for establishing neuronal polarity, and BRSK1 has also been shown to regulate neurotransmitter release presynaptically. How BRSK1 exerts this latter function is unknown, since its substrates at the synaptic terminal and the mechanisms modulating its activity remain to be described. Key regulators of neurotransmitter release, such as SNARE complex proteins, are located at membrane rafts. Therefore we initially undertook this work to check whether BRSK1 also locates at these membrane microdomains. Here we show that brain BRSK1, but not BRSK2, is palmitoylated, and provide biochemical and pharmacological evidences demonstrating that a pool of BRSK1, but not BRSK2 or LKB1, localizes at membrane lipid rafts. We also show that raft-associated BRSK1 has higher activity than BRSK1 from non-raft environment, based on a higher T-loop phosphorylation at Thr-189. Further, recombinant BRSK1 activity increased 3-fold when assayed with small multilamellar vesicles (SMV) generated with lipids extracted from synaptosomal raft fractions. A similar BRSK1-activating effect was obtained with synthetic SMV made with phosphatidylcholine, cholesterol and sphingomyelin, mixed in the same molar ratio at which these three major lipids are present in rafts. Importantly, SMV also enhanced the activity of a constitutively active BRSK1 (T189E), underpinning that interaction with lipid rafts represents a new mechanism of BRSK1 activity modulation, additional to T-loop phosphorylation.► A pool of brain BRSK1, but not BRSK2 or LKB1, localizes at membrane lipid rafts. ► Brain BRSK1 is palmitoylated. ► Raft-associated BRSK1 has higher activity than BRSK1 from non-raft environment. ► Small multilamellar vesicles made with lipids from rafts enhance BRSK1 activity. ► BRSK1-raft interaction: a new activating mechanism additional to LKB1 phosphorylation.
Keywords: BRSK1; SAD kinase; LKB1; Lipid raft; Palmitoylation; Small multilamellar vesicle;

Synthetic LXR agonist inhibits the development of atherosclerosis in New Zealand White rabbits by Shoko Honzumi; Akiko Shima; Ayano Hiroshima; Tadashi Koieyama; Naoki Terasaka (1136-1145).
The nuclear receptors Liver X receptors, LXRα and LXRβ, regulate cholesterol and triglyceride metabolism. We and others have previously reported that synthetic LXR agonists reduced atherosclerosis in models of mouse with no detectable plasma cholesteryl ester transfer protein (CETP) activity, which plays an important role in reverse cholesterol transport. In the present study, we investigated the effect of LXR activation in rabbits to elucidate the influence of CETP activity. First, we cloned rabbit LXRs cDNA. The data indicated that rabbit LXRα was mostly highly expressed in the liver, whereas LXRβ expression was ubiquitous. Next, we investigated the effect of LXR agonist on lipid levels. Treatment with LXR agonist T0901317 increased plasma CETP activity and consequently elevated LDL, but no change in HDL. High cholesterol (HC) diet-feeding, which is thought to provide oxysterols as the natural agonists, could also increase expression of CETP and other LXR target genes. Finally, we tested T0901317 in the atherosclerosis intervention study. Chronic administration of T0901317 significantly reduced atherosclerosis in HC diet-fed rabbits despite less favorable lipid profiles, i.e. increases of plasma triglycerides and no change of HDL. T0901317 induced ATP-binding cassette transporters ABCA1 and ABCG1 and suppressed inflammatory genes expression in the aorta, suggesting that direct actions of LXR agonist on vascular gene expression are likely to contribute to the antiatherogenic effect. The present work strongly supports the idea that LXR agonists could be beneficial as therapeutic agents for treatment of atherosclerosis.► We cloned rabbit LXRs cDNA. ► LXR activation in rabbits affect plasma lipid profiles in a different manner in rodents with no CETP activity. ► LXR agonist reduced atherosclerosis in rabbits, suggesting direct antiatherogenic effects mediated by induction of vascular gene expression.
Keywords: CETP; Cloning; HDL; LXR; Rabbit; Atherosclerosis;

Estrogen upregulates hepatic apolipoprotein M expression via the estrogen receptor by Jiang Wei; Yuanping Shi; Xiaoying Zhang; Yuehua Feng; Guanghua Luo; Jun Zhang; Qinfeng Mu; Yanhong Tang; Yang Yu; Lili Pan; Peter Nilsson-Ehle; Ning Xu (1146-1151).
Apolipoprotein M (apoM) is present predominantly in high-density lipoprotein (HDL) in human plasma, thus possibly involved in the regulation of HDL metabolism and the process of atherosclerosis. Although estrogen replacement therapy increases serum levels of apoAI and HDL, it does not seem to reduce the cardiovascular risk in postmenopausal women. Therefore, we investigated the effects of estrogen on apoM expression in vitro and in vivo. HepG2 cells were incubated with different concentrations of estrogen with or without the estrogen receptor antagonist, fulvestrant, and apoM expression in the cells was determined. Hepatic apoM expression and serum levels of apoM were also determined in normal and in ovariectomized rats treated with either placebo or estradiol benzoate, using sham operated rats as controls. Estrogen significantly increased mRNA levels of apoM and apoAI in HepG2 cell cultures in a dose- and time-dependent manner; the upregulation of both apolipoproteins was fully abolished by addition of estrogen receptor antagonist. In normal rats, estrogen treatment led to an increase in plasma lipid levels including HDL cholesterol, a marked upregulation of apoM mRNA and a significant increase in serum levels of apoM. The same pattern of regulation was found in ovariectomized rats treated with estrogen. Thus, estrogen upregulates apoM expression both in vivo and in vitro by mechanism(s) involving the estrogen receptor.► It clearly demonstrated that estrogen upregulates both apoAI and apoM expressions. ► Estrogen enhanced apoM expression with a dose- and time-dependent manner in cells. ► This up-regulative effect is mediated via the estrogen receptor. ► The cellular observations were further confirmed in animal models.
Keywords: Apolipoprotein M; HDL; Estrogen; Lipid metabolism;

The ATP-binding cassette transporter-2 (ABCA2) has been identified as a possible regulator of lipid metabolism. ABCA2 is most highly expressed in the brain but its effects on cholesterol homeostasis in neuronal-type cells have not been characterized. It is important to study the role of ABCA2 in regulating cholesterol homeostasis in neuronal-type cells because ABCA2 has been identified as a possible genetic risk factor for Alzheimer's disease. In this study, the effects of ABCA2 expression on cholesterol homeostasis were examined in mouse N2a neuroblastoma cells. ABCA2 reduced total, free- and esterified cholesterol levels as well as membrane cholesterol but did not perturb cholesterol distribution in organelle or lipid raft compartments. ABCA2 did not modulate de novo cholesterol biosynthesis from acetate. Cholesterol trafficking to the plasma membrane was not affected by ABCA2 but efflux to the physiological acceptor ApoE3 and mobilization of plasma membrane cholesterol to the endoplasmic reticulum for esterification were reduced by ABCA2. ABCA2 reduced esterification of serum and low-density lipoprotein-derived cholesterol but not 25-hydroxycholesterol. ABCA2 decreased low-density lipoprotein receptor (LDLR) mRNA and protein levels and increased its turnover rate. The surface expression of LDLR as well as the uptake of fluroresecent DiI-LDL was also reduced by ABCA2. Reduction of endogenous ABCA2 expression by RNAi treatment of N2a cells and rat primary cortical neurons produced the opposite effects of over-expression of ABCA2, increasing LDLR protein levels. This report identifies ABCA2 as a key regulator of cholesterol homeostasis and LDLR metabolism in neuronal cells.► ABCA2 expression reduced membrane, total, free- and cholesterol ester levels. ► ABCA2 expression reduced cholesterol efflux to cyclodextrin and ApoE3. ► ABCA2 expression reduced esterification low-density lipoprotein-derived cholesterol. ► ABCA2 expression reduced low-density lipoprotein receptor (LDLR) levels. ► Silencing of endogenous ABCA2 expression increased LDLR protein levels.
Keywords: ATP-binding cassette transporter; ABCA2; Cholesterol; Low-density lipoprotein receptor; LDLR;

Lipid particles/droplets of the yeast Saccharomyces cerevisiae revisited: Lipidome meets Proteome by Karlheinz Grillitsch; Melanie Connerth; Harald Köfeler; Tabiwang N. Arrey; Benjamin Rietschel; Brigitte Wagner; Michael Karas; Günther Daum (1165-1176).
In the yeast Saccharomyces cerevisiae as in other eukaryotes non-polar lipids are a reservoir of energy and building blocks for membrane lipid synthesis. The yeast non-polar lipids, triacylglycerols (TG) and steryl esters (SE) are stored in so-called lipid particles/droplets (LP) as biologically inert form of fatty acids and sterols. To understand LP structure and function in more detail we investigated the molecular equipment of this compartment making use of mass spectrometric analysis of lipids (TG, SE, phospholipids) and proteins. We addressed the question whether or not lipid and protein composition of LP influence each other and performed analyses of LP from cells grown on two different carbon sources, glucose and oleate. Growth of cells on oleate caused dramatic cellular changes including accumulation of TG at the expense of SE, enhanced the amount of glycerophospholipids and strongly increased the degree of unsaturation in all lipid classes. Most interestingly, oleate as a carbon source led to adaptation of the LP proteome resulting in the appearance of several novel LP proteins. Localization of these new LP proteins was confirmed by cell fractionation. Proteomes of LP variants from cells grown on glucose or oleate, respectively, were compared and are discussed with emphasis on the different groups of proteins detected through this analysis. In summary, we demonstrate flexibility of the yeast LP lipidome and proteome and the ability of LP to adapt to environmental changes.► Molecular analysis of yeast lipid particles/droplets components. ► Combination of lipidome and proteome analysis. ► Effect of oleate stress on lipid particle lipidome and proteome. ► High flexibility of lipid particles on environmental conditions.
Keywords: Triacylglycerol; Phospholipid; Mass spectrometry; Protein; Lipid particle/droplet; Yeast;

Phosphatidylcholine protects against steatosis in mice but not non-alcoholic steatohepatitis by Lorissa J. Niebergall; René L. Jacobs; Todd Chaba; Dennis E. Vance (1177-1185).
Several studies suggest that low levels of hepatic phosphatidylcholine (PC) play a role in the pathogenesis of non-alcoholic steatohepatitis (NASH). CTP: phosphocholine cytidylyltransferase (CT) is the key regulatory enzyme in the CDP-choline pathway for PC biosynthesis. Liver-specific elimination of CTα (LCTα−/−) in mice fed a chow diet decreases very-low-density lipoprotein secretion, reduces lipid efflux from liver, and causes mild steatosis. We fed LCTα−/− mice a high fat diet to determine if impaired PC biosynthesis played a role in development of NASH. LCTα−/− mice developed NASH within one week of high fat feeding. Hepatic CTα deficiency caused hepatic steatosis, a 2-fold increase in ceramide mass, and a 20% reduction in PC content. In an attempt to prevent NASH, LCTα−/− mice were either injected daily with CDP-choline or fed the high fat diet supplemented with betaine. In addition, LCTα−/− mice were injected with adenoviruses expressing CTα. CDP-choline injections and adenoviral expression of CTα increased hepatic PC, while dietary betaine supplementation normalized hepatic triacylglycerol but did not alter hepatic PC mass in LCTα−/− mice. Interestingly, none of the treatments normalized hepatic ceramide mass or fully prevented the development of NASH in LCTα−/− mice. These results show that normalizing the amount of hepatic PC is not sufficient to prevent NASH in LCTα−/− mice.►Hepatic CTα knockout mice are susceptible to high fat diet-induced liver NASH. ►Hepatic CTα knockout mice have 25% lower hepatic PC resulting in steatosis. ►Diacylglycerol and ceramide are elevated by 50% in CTα-deficient livers. ►Normalization of hepatic PC prevents steatosis in hepatic CTα knockout mice. ►Hepatic level of PC is not the only determinant for the development of NASH.
Keywords: Betaine; CDP-choline; Diacylglycerol; Liver; Ceramide;

ATP-binding cassette G5/G8 deficiency causes hypertriglyceridemia by affecting multiple metabolic pathways by Jesús Méndez-González; Josep Julve; Noemí Rotllan; Gemma Llaverias; Francisco Blanco-Vaca; Joan Carles Escolà-Gil (1186-1193).
Mutations in ABCG5 or ABCG8 transporters are responsible for sitosterolemia, an autosomal recessive disease characterized by the accumulation of plant sterols. The aim of this study was to investigate the effects of ABCG5 and ABCG8 deficiency on TG metabolism in mice. Experiments were carried out in wild-type (G5/G8+/+) mice, mice heterozygous for ABCG5 and ABCG8 deficiency (G5/G8+/−) and ABCG5/G8-deficient (G5/G8−/−) mice fed a chow diet. Plasma TG were 2.6 and 4.3-fold higher in fasted G5/G8+/− and G5/G8−/− mice, respectively, than in G5/G8+/+ mice. Postprandial TG were 5-fold higher in G5/G8−/− mice. TG metabolism studies indicate that: first, the fractional catabolic rate was significantly lower in G5/G8+/− (1.3-fold) and G5/G8−/− mice (1.5-fold) compared to G5/G8+/+ and postheparin plasma lipoprotein lipase activities were significantly lower in G5/G8+/− (1.8-fold) and G5/G8−/− mice (5.4-fold) than in G5/G8+/+. Second, liver TG secretion was 1.3-fold higher in G5/G8+/− and G5/G8−/− than in G5/G8+/+ mice and this was associated with an increase in liver LXR, FAS, ACAC and CD36 gene expression. Third, TG intestinal secretion, determined after an oral fat gavage of glycerol tri[9,10(n)-3H] oleate, was 5.8-fold higher in G5/G8−/− than in G5/G8+/+ mice. Also, the HOMA index was 2.6-fold higher in G5/G8−/− than in G5/G8+/+ mice, reflecting a degree of insulin resistance. In conclusion, ABCG5/G8 deficiency in mice fed a chow diet markedly raises TG levels by impairing TG catabolism and by increasing liver and intestinal TG secretion.► ABCG5/G8 deficiency increases both fasting and postprandial plasma TG levels. ► ABCG5/G8 deficiency impairs TG catabolism by lowering LPL activity. ► ABCG5/G8 deficiency increases both liver and small intestinal TG secretion.
Keywords: ABCG5 and ABCG8; Lipoprotein lipase; Triglyceride; Liver X receptor; Very-low-density lipoprotein;

ChREBP expression in the liver, adipose tissue and differentiated preadipocytes in human obesity by Carmen Hurtado del Pozo; Gregorio Vesperinas-García; Miguel-Ángel Rubio; Ramón Corripio-Sánchez; Antonio J. Torres-García; Maria-Jesus Obregon; Rosa María Calvo (1194-1200).
ChREBP is an essential transcription factor for lipogenesis. Its physiological role in adipose tissue has been studied only to a small extent and the control of its expression remains unknown in human adipocytes. We have studied ChREBP mRNA and protein expression levels in the liver and the omental (OM) and subcutaneous (SC) adipose tissues from obese and lean subjects, as well as in human differentiated preadipocytes. Liver and OM and SC adipose tissue biopsies were obtained from lean and obese patients. Human preadipocytes were isolated from the adipose tissues from obese patients and differentiated under adipogenic conditions. ChREBP expression levels were quantified by RT-PCR and Western blot analysis. We found opposing results in terms of ChREBP regulation in the liver and adipose samples. ChREBP increased in the liver from obese compared to lean subjects, whereas the expression decreased in both adipose tissues. The mRNAs of other adipogenic markers were checked in these tissues. The pattern of FASN was similar to the one for ChREBP, ADCY3 decreased in both adipose tissues from obese patients, AP2 decreased only in OM adipose tissue of obese patients and ATGL did not change. The levels of ChREBP mRNA and protein showed dramatic increases during the differentiation of human OM and SC preadipocytes. In conclusion, ChREBP expression has an opposite regulation in the liver and adipose tissue from obese subjects which is compatible with the increased hepatic lipogenesis and decreased adipocytic lipogenesis found in these patients. The dramatic increase of ChREBP mRNA and protein levels during preadipocyte differentiation suggests a role in adipogenesis.► ChREBP was studied in human liver and adipose tissues in lean and obese patients. ► ChREBP mRNA and protein increased in the liver from obese patients. ► ChREBP mRNA levels in both adipose tissues decreased in obese individuals. ► Similar changes in the lipogenic enzyme FASN were observed in these tissues. ► ChREBP increases during differentiation of human preadipocytes in vitro.
Keywords: ChREBP; Human liver; Subcutaneous adipose tissue; Omental; Preadipocyte;