European Network
for Oxysterol Research
3rd ENOR symposium
“OXYSTEROLS: Markers and Pathways”
Swansea - United Kingdom
19-20 September 2013
Dear participants, welcome to Swansea !
The ENOR meeting series was started with successful by our colleagues
successively from Munich in 2010, from Roma in 2011 and from Dijon in 2012.
As defined in 2010 the main goals of the ENOR meeting are :
 to provide an international forum for researchers to present their results of
ongoing research (oral presentations, posters),
 to bring European oxysterols researchers together,
 to stimulate discussions and possible collaborations.
It is a great pleasure to receive you and your co-workers at the University of
Swansea for this 3rd European Network on Oxysterols Research symposium.
Organizing Committee
William Griffiths
University of Swansea, UK
Yuqin Wang
University of Swansea, UK
Gérard Lizard
University of Burgundy, France
Peter Crick
University of Swansea, UK
Luigi Iuliano
University of Rome, Italy
1
Index
Program ...............................................................................................................................p 5
Plenary lecture : Professor Jan-Ǻke Gustafsson, University of Houston ...........................p 8
Abstracts
Paolo Abrescia, University of Naples, Italy .........................................................................p 9
Ruth Andrew, University of Edinburgh, United of Kingdom ............................................p 10
ErnestArenas, Karoslinska Institute of Stockholm, Sweden .............................................p 11
Silvère Baron, Université Blaise Pascal Clermont-Ferrand, France ..................................p 12
Ingemar Björkhem, Karolinska Institut of Stockholm, Sweden ........................................p 13
Sérgio Paulo Bydlowski, University of São Paulo, Brazil ..................................................p 14
Vladimir Cardenia, University of Bologna, Italy................................................................p 15
Peter J. Crick, Swansea University, United of Kingdom ....................................................p 16
Ulf Diczfalusy, Karolinska Institute of Stockholm, Sweden ..............................................p 17
Dieter Lütjohann, University of Bonn, Germany .............................................................p 18
Peter Ghazal, University of Edinburgh, United of Kingdom ............................................p 19
Catherine Gondcaille, University of Burgundy, France ...................................................p 20
Youssef El Kharrass, University of Burgundy, France .......................................................p 21
Valerio Leoni, Institutes of Neurology Carlo Besta, Italy ..................................................p 22
Salwan Magdasy, University of Clermonf-Ferrand, France ..............................................p 23
S. Matysik, University Hospital Regensburg, Germany ...................................................p 24
Noriyuki Miyoshi, University of Shizuoka, Japan .............................................................p 25
Noriko Noguchi ................................................................................................................p 26
Thomas Nury, Universiry of Burgugndy, France ..............................................................p 27
Vesa M. Olkkonen, Institute for Medical Research Helsinki, Finland .............................p 28
Marc Poirot, Cancer Research Center of Toulouse, France .............................................p 29
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3rd ENOR symposium – Swansea 2013
K. Rigdova, Swansea University, United of Kingdom .......................................................p 30
Hanne Røberg-Larsen, University of Oslo, Norway .........................................................p 31
Kevin A. Robertson, University of Edinburgh, United of Kingdom ..................................p 32
Maria Theresa Rodriguez-Estrada, University of Bologna, Italy .....................................p 33
Andreas W. Sailer, Novartis Institutes Basel, Switzerland ..............................................p 34
Yoshiro Saito, Doshiha University, Japan .........................................................................p 35
Zuzana Simova, Swansea University, United og Kingdom ...............................................p 36
Spyridon Theofilopoulos, Karolinska Institute of Stockholm, Sweden ............................p 37
Vaya Jacod, Tel-Hai College, Israel ...................................................................................P 39
Oliver Weingärtner, University of Bonn, Germany ..........................................................p 40
Wei Yuan Hsieh, University of Edinburgh, United of Kingdom ........................................p 41
Steven Ray Wilson, University of Oslo, Norway ...............................................................p 42
Author index ......................................................................................................................p 44
Sponsors ............................................................................................................................p 47
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3rd ENOR symposium – Swansea 2013
Program
3 rd ENOR Symposium
Oxysterols: Markers and Pathways
Swansea, UK. 19-20 September 2013
Plenary lecture
Jan-Ǻke Gustafsson, University of Houston. “Liver X receptors – multifunctional ligand
activated transcription factors”.
Session 1. Oxysterols and nuclear receptors
Ernest Arenas, Karolinska Institutet, “Identification of novel brain endogenous Liver X
Receptor ligands that selectively promote red nucleus or midbrain dopamine
neurogenesis”.
Salwan Maqdasy, Clermont Université, “Oxysterols, Liver X Receptors and male
reproduction”.
Marc Poirot, University Toulouse III, “Discovery of Dendrogenin A as a product of
cholesterol and histamine metabolism with cell differentiation and anti-tumour
properties”.
Session 2. Oxysterol binding proteins and enzymes
Vesa M. Olkkonen, Minerva Foundation Institute for Medical Research, “Oxysterolbinding proteins: Sterol and phosphoinositide sensors with versatile functions in cell
regulation”.
Ulf Diczfalusy, Karolinska Institutet, “On the formation of 7-ketocholesterol”.
Session 3. Oxysterols and the nervous system
Ingemar Björkhem, Karolinska Institutet, “Is it possible to improve memory function by
upregulation of the cholesterol 24S-hydroxylase (CYP46A1) in the brain?”.
Valerio Leoni, Foundation IRCCS Institute of Neurology Carlo Besta, “Impairment of
cholesterol metabolism in Huntington Diseases: evidence of anabolic dysfunction in
neurodegenerative process”.
Noriko Noguchi, Doshisha University, “Diverse functions of 24(S)-hydroxycholesterol
in the brain”.
Paolo Abrescia, University of Naples Federico II, “Does LCAT play a role in limiting
the cerebrosterol neurotoxicity?”.
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3rd ENOR symposium – Swansea 2013
Spyridon Theofilopoulos, Karolinska Institutet, “Identification of Cholestenoic acids
altered in motor neuron patients that regulate motor neuron survival via Liver X
Receptors”.
Session 4. Oxysterols and immunity
Peter Ghazal, University of Edinburgh, “Macrophages, viruses and Ch25h”.
Andreas W. Sailer, Novartis Institutes for BioMedical Research, “Signaling of the
oxysterol / EBI2 pathway in immune cells”.
Kevin A. Robertson, University of Edinburgh, “microRNA targeting SREBP2 and
coupling the antiviral interferon response to the regulation of cholesterol biosynthesis”.
Session 5. Biological activity of oxysterols
Sérgio Paulo Bydlowski, University of São Paulo School of Medicine, “Oxysterols
induce dose-dependent cell proliferation or death in human mesenchymal stem cells
(MSC) in vitro”.
Gérard Lizard, Université de Bourgogne, “Induction of a dual mode of cell death
(autophagy
and
apoptosis)
on
7-ketocholesterol-treated
158N
murine
oligodendrocytes”.
Noriyuki Miyoshi, University of Shizuoka, “Cholesterol aldehydes: The mechanisms of
their formation and pathological roles in oxidative stress related-disorders”.
Session 6. Oxysterol and sterols as biomarkers
Yoshiro Saito, Doshisha University, “7-Hydroxycholestrol as a possible biomarker of
cellular lipid peroxidation- difference between cellular and plasma lipid peroxidation”.
Vaya Jacob, Tel-Hai College, “Lipids, oxidized lipids and proteins levels among
Symptomatic versus Asymptomatic”.
Oliver Weingärtner, “Paradoxical increase in cholesterol concentrations in serum and
in circulating monocytes by plant sterol ester supplemented margarine. A prospective,
double-blind, randomized, placebo-controlled, cross-over study.”
Iuliano Luigi, Sapienza University, “Oxysterol profiling in seminal fluid: is there any
connection with infertility?”.
Session 7. Sterol and oxysterol analysis
S. Matysik, University Hospital Regensburg, “GC-QToF with atmospheric chemical
ionization - a new approach to detect oxysterols”.
Steven Ray Wilson, University of Oslo, “Oxysterols and the Hedgehog signaling
pathway”.
Ruth Andrew, University of Edinburgh, “Quantifying keto-steroids in brain by Mass
Spectrometry Imaging with on-tissue derivatisation”.
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3rd ENOR symposium – Swansea 2013
William J. Griffiths, Swansea University, “Low-level analysis of oxysterols in cells,
tissues and fluids”.
Dieter Lütjohann, University of Bonn, “First worldwide non-cholesterol and oxysterol
survey”.
Session 8. Sponsors talks
Therese Koal, BIOCRATES Life Sciences AG.
Thermo Fisher
Session 9. Posters
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3rd ENOR symposium – Swansea 2013
Plenary lecture
Professor Jan-Ǻke Gustafsson, University of Houston
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3rd ENOR symposium – Swansea 2013
Abstracts
Does LCAT play a role in limiting the cerebrosterol neurotoxicity?
Valeria La Marca1, Fabrizio Dal Piaz2 and Paolo Abrescia1#
1
University of Naples Federico II – Dept Biology, via Mezzocannone 8 – 80134 Naples, Italy.
University of Salerno, Dept Pharmacy, via Giovanni Paolo II 132 – 84084 Fisciano (Salerno),
Italy.
# paolo.abrescia@unina.it
2
The esterification of cerebrosterol (24OH-C) was suggested to occur in the CNS for
enhancing the 24OH-C transport through the brain-blood barrier. The enzyme LCAT, known
to esterify cholesterol and a number of oxysterol, was hypothesized to convert 24OH-C into
cerebrosteryl esters (24OH-CE). We report here that LCAT effectively esterifies 24OH-C in
vitro, under stimulation by ApoA-I or ApoE. Proteoliposomes containing fluorescent
phospholipids and either apolipoprotein were used to synthesize 24OH-C monoesters for
mass spectrometry analysis. 24OH-CE were also synthesized from tritium-labelled 24OH-C
and proteoliposomes with natural phosphatidylcholine, and the Km values for the enzyme
activity with ApoA-I and ApoE were determined. 24OH-C was found to compete with
cholesterol for the LCAT activity. This activity was impaired by high levels of haptoglobin, and
oxidants such as Cu+2/H2O2 or peroxynitrite. The survival of SH-SY5Y cells in culture was
severely impaired by 24OH-C, yet significantly improved when 24OH-C was previously
incubated with LCAT and proteoliposomes. Proteoliposome-embedded 24(S)OH-CE did not
enter SH-SY5Y cells, thus suggesting that the enzyme might restrict 24OH-C to the brain
extracellular environment, by converting unesterified into ester form for transport across
the brain-blood barrier toward the circulation. The LCAT activity in CNS, expressed as 24OHCE/24OH-C ratio, was found dramatically decreased in cerebrospinal fluid from patients with
Alzheimer’s disease. This ratio was also calculated in the patients’ plasma, and the obtained
values well correlated with those found in homologous cerebrospinal fluids. A high number
of samples, particularly from subjects with mild cognitive impairment, must be analysed to
assess whether the LCAT activity in CNS is a new therapeutic target, and can be used as a
biomarker for diagnosing prodromal Alzheimer’s disease or evaluating the patient response
to treatment.
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3rd ENOR symposium – Swansea 2013
Quantifying keto-steroids in brain by Mass Spectrometry Imaging
with on-tissue derivatisation
Ruth Andrew
Endocrinology, Queen's Medical Research Institute, University of Edinburgh, 47, Little France
Crescent, Edinburgh, EH16 4TJ
Intracellular enzymatic inter-conversion of steroids influences receptor activation, providing
therapeutic targets. However, inadequate methods exist to assess steroids in constituent
cells within tissues. We have previously developed a method to detect keto-steroids in
regions of tissue sections using derivatisation. Here, mass spectrometry imaging (MSI) was
used to quantitatively profile inert and active glucocorticoids (11-dehydrocorticosterone,
corticosterone), the substrates and products of 11β-hydroxysteroid dehydrogenase 1
(11βHSD1) respectively
Mice with genetic disruption of 11βHSD1 (11HSD1-/- vs wild-type) and control mice
receiving a pharmacological inhibitor of 11βHSD1 (or vehicle) were studied (n=6/group).
Steroids were imaged in brain sections by Matrix Assisted Laser Desorption Ionization,
Fourier transform Ion cyclotron MS, following on-tissue derivatisation with Girard T.
Quantitation was confirmed by LC-MS/MS and steroids unambiguously identified by Liquid
Extraction Surface Analysis (with collision induced dissociation; LESA); *p<0.05
The ratio of corticosterone/11-dehydrocorticosterone was reduced in 11βHSD1 deficiency
(% wild-type; cortex 50%*; hippocampus 45%*; amygdala 58 %*) and following 11β-HSD1
inhibition, (% vehicle; cortex 22%*; hippocampus 25%*; amygdala 36 %*). LESA confirmed
identities of ions and quantitative data correlated excellently with measurements by LCMS/MS.
MSI with derivatization is a powerful new tool to localize and quantify small lipophilic
molecules, such as steroids regionally within tissues. Opportunities exist to translate the
method to other keto-steroids and keto-oxysterols.
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3rd ENOR symposium – Swansea 2013
Identification of novel brain endogenous Liver X Receptor ligands that
selectively promote red nucleus or midbrain dopamine neurogenesis
Spyridon Theofilopoulos1, Yuqin Wang2, Satish Srinivas Kitambi1,
Paola Sacchetti1,7, Kyle MSousa1,7, Karl Bodin1, Jayne Kirk3, Carmen Saltó1,
Magnus Gustafsson1, Enrique MToledo1, Kersti Karu4, Jan-Åke Gustafsson5,
Knut RSteffensen6, Patrik Ernfors1, Jan Sjövall1, William J Griffiths2
and Ernest Arenas1
1
Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and
Biophysics, Karolinska Institute, Stockholm, Sweden.
2 Institute of Mass
Spectrometry, College of Medicine, Swansea university, Singleton Park, Swansea, U.K.
3 Waters UK ltd., Manchester, UK.
4 The School of Pharmacy, London, UK.
5 Center for Nuclear receptors and Cell Signaling, university of Houston, Houston, Texas, USA
6 Department of biosciences and Nutrition, Center for biosciences, Stockholm, Sweden.
7 Present addresses: Department of biological Sciences, Mount Holyoke College, South
Hadley, Massachusetts, USA (P.S.);
Department of Gene regulation and Drug Discovery, City of Hope–beckman research
Institute, Duarte, California, USA (K.M.S.).
Liver X receptors (Lxrα and Lxrβ) are ligand-dependent nuclear receptors critical for ventral
midbrain neurogenesis in vivo. However, no endogenous midbrain Lxr ligand has so far been
identified. Here we used LC/MS and functional assays to identify cholic acid as a new Lxr
ligand. Moreover, 24(S),25-epoxycholesterol (24,25-EC) was found to be the most potent
and abundant Lxr ligand in the developing mouse midbrain. Both Lxr ligands promoted
neural development in an Lxr-dependent manner in zebrafish in vivo. Notably, each ligand
selectively regulated the development of distinct midbrain neuronal populations. Whereas
cholic acid increased survival and neurogenesis of Brn3a-positive red nucleus neurons,
24,25-EC promoted dopaminergic neurogenesis. These results identify an entirely new class
of highly selective and cell type–specific regulators of neurogenesis and neuronal survival.
Moreover, 24,25-EC promoted dopaminergic differentiation of embryonic stem cells,
suggesting that Lxr ligands may thus contribute to the development of cell replacement and
regenerative therapies for Parkinson's disease.
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3rd ENOR symposium – Swansea 2013
LXR null mice, a promising model to decipher connection between cholesterol
metabolism and prostate cancer.
Aurélien J.C. Pommier1,2,3,4, Julie Dufour1,2,3,4, Georges Alves1,2,3,4,
Emilie Viennois1,2,3,4, Hugues De Boussac1,2,3,4, Amalia Trousson1,2,3,4,
David H. Volle1,2,3,4, Françoise Caira1,2,3,4, Pierre Val1,2,3, Philippe Arnaud1,2,3,
Jean-Marc A. Lobaccaro1,2,3,4 and Silvère Baron1,2,3,4
1Clermont
université, Université Blaise Pascal, Génétique Reproduction et Développement, BP
10448, F63000 CLERMONT-FERRAND, France
2CNRS, UMR 6293, GReD, F-63177 AUBIERE, France
3INSERM, UMR 1103, GReD, F-63177 AUBIERE, France
4Centre de Recherche en Nutrition Humaine d’Auvergne, F-63000 CLERMONT-FERRAND,
France
Liver X Receptors belong to the nuclear receptor superfamily and play a central role in
cholesterol homeostasis. Increasing studies pointed out that LXRs could be considered
promising therapeutic targets in prostate cancer. Present work investigates the prostatic
phenotype of LXR null mice and characterizes molecular features of these animals.
Phenotypic exploration and microarray analysis have been performed on prostate from LXR
null or wild type mice fed a normal or a high cholesterol diet.
LXR-/- mice fed a high cholesterol diet harbor abnormal cholesterol esters accumulation
associate with PIN lesion development. Microarray analysis reveals a signature of prostate
cancer characterized by the deregulation of key tumor suppressor genes such as nkx3.1 and
msmb. Functional analysis and ChIP experiments have demonstrated that the upstream
regulator EZH2 was also deregulated. This strongly suggests that epigenetic process is
disturbed by metabolic changes in prostatic cells.
Finally, this study provide evidences that LXRs are a major regulator of prostatic cell
metabolism and that potential impairment of their expression and/or function could be
relevant in human prostate cancer initiation and progression.
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3rd ENOR symposium – Swansea 2013
Is it possible to improve memory function by upregulation of the cholesterol
24S-hydroxylase (CYP46A1) in the brain?
Ingemar Björkhem, Silvia Maioli, Ann Båvner and Angel Cedazo-Minguez
Department of Laboratory Medicine, Division of Clinical Chemistry and Department of
Neurobiology, Care Sciences and Society, Karolinska Institut of Stockholm, Sweden
Cholesterol 24S-hydroxylase (CYP46) is critical for brain cholesterol homeostasis. In contrast
to cholesterol itself 24S-hydroxycholesterol (24OH) is able to pass the blood-brain barrier
and reach the circulation. In addition CYP46 seems to be of importance for memory. David
Russell and his group demonstrated that a knock-out of CYP46 in mice resulted in severe
deficiencies in spatial, associative and motor learning. These changes were associated with
electrophysiological changes in hippocampal slices. Treatment of such slices of wild type
mice with an inhibitor of cholesterol synthesis recapitulated the effects observed in cyp46-/mice. Both the genetic and the pharmacological effects could be reversed by addition of
geranylgeraniol. It was hypothesized that a continuous formation of 24OH is needed for
sufficient flux through the mevalonate pathway to generate adequate levels of
geranylgeraniol. Geranylation was suggested to be important for the function of some
proteins critical for memory function.
In the present work we have tested the hypothesis that an increased activity of CYP46 with
increased consumption of cholesterol in the mevalonate pathway may improve memory
functions in mice.
We generated a mouse model overexpressing human 24S-hydroxylase (CYP46A1) utilizing a
ubiquitous expression vector. Female homozygous CYP46A1 transgenic mice, aged 15
months, showed an improvement in spatial memory in the Morris water maze test as
compared to the wild type mice. The levels of N-Methyl-D-Aspartate receptor 1,
phosphorylated-N-Methyl-D-Aspartate receptor 2A, postsynaptic density 95, synapsin-1 and
synapthophysin were significantly increased in the hippocampus of the CYP46A1 transgenic
mice as compared to the controls. The levels of lanosterol in the brain of the CYP46A1
transgenic mice were significantly increased, consistent with a higher synthesis of
cholesterol.
Our results are discussed in relation to the hypothesis that the flux in the mevalonate
pathway in the brain is of importance for cognitive functions.
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3rd ENOR symposium – Swansea 2013
Oxysterols induce dose-dependent cell proliferation or death in human
mesenchymal stem cells (MSC) in vitro
Jorge Luis M. Ruiz, Andrea Turbuck Celestino, Débora Levy, Ana Carolina Bazan
de Carvalho, Suelen Feitoza Silva and Sérgio Paulo Bydlowski
Laboratory of Genetics and Molecular Hematology (LIM31), University of São Paulo School of
Medicine, Av. Dr. Enéas de Carvalho Aguiar, 155, 1st Floor, Room 43, 05403-000 São
Paulo/SP, Brazil
Mesenchimal stem cells (MSCs) are multipotent stromal cells with self-renewal and
differentiation potential. One of the few reports on the role of oxysterols in the homeostasis
of stem cell physiology showed that some oxysterols could affect the Hedgehog signaling
pathway.
To better understand the effect of oxysterols in MSCs, MSCs were isolated from human
adipose tissue (hATMSCs), human amniotic fluid (hAFMSCs) and human Wharton’s jelly
(hWJMSCs). The effect of 7-ketocholesterol (7KC), cholestan 3-beta 5-alpha 6-beta-triol
(Triol) and cholesterol (Chol) in MSCs proliferation and death was examined.
MSCs were characterized by 1. expression of undifferentiation markers (Oct-4, Sox-2, Nanog)
by RT-PCR, 2. identification of cell surface proteins by flow cytometry, and 3. cell
differentiation assays into adipogenic, chondrogenic and osteogenic lineages, confirmed
with Alizarin Red, H&E and Oil red O stains, respectively. Effects of several concentrations of
7KC, Triol and Chol (0.01 to 100uM) on MSCs proliferation and death were evaluated by
MTT assay after 24 h incubation. Fibroblasts (MRC-5 cells;ATCC: CCL-171) were used as
control. Data were evaluated by curve-fitting with Prism 5.0 software (GraphPad, San Diego,
CA).
The presence of MSCs in all experiments was confirmed by the expression of pluripotent
stem cell specific genes (Oct-4, Sox-2 and nanog) and by the expression of mesenchymal
membrane markers (CD29+, CD44+ and CD133+). The cells were also capable to differentiate
into distinct lineages: adipocyte, osteoblasts and chondrocytes.
Cholesterol did not affect MSC proliferation or death, at least in the used concentrations.
7KC and Triol increased hAFMSCs death as well as fibroblast death with concentrations
higher than 10µM in a dose-dependent manner. The same results were obtained with
hATMSCs and hWJMSCs when Triol was added in a concentration higher than 1µM, also in a
dose-dependent way. Interestingly, 7KC (concentrations from 1 and 10µM) led to cell
proliferation in all studied MSCs. This effect was not observed in fibroblasts. Moreover, at
least after 24 h incubation, no morphological changes were observed.
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3rd ENOR symposium – Swansea 2013
Volatile compounds as rapid screening markers of cholesterol oxidation
Vladimiro Cardenia1 and Maria Teresa Rodriguez-Estrada2
1
Interdepartmental Centre for Agri-food Industrial Research, Alma Mater StudiorumUniversity of Bologna (Italy);
2 Department of Agricultural and Food Sciences, Alma Mater Studiorum-University of Bologna
(Italy)
Cholesterol oxidation products (COPs) are widely studied because they are likely involved in
various chronic and degenerative diseases. However, COPs determination is an expensive,
time-consuming analysis, as it requires long preparative steps (extraction of unsaponifiable
matter, COPs isolation by SPE, derivatization), and high-efficiency and sensitivity equipment
(such as mass spectrometry). The aim of this work was to develop a cheaper and rapid
screening of cholesterol oxidation by SPME-GC/MS analysis of volatile compounds.
Cholesterol was thermoxidized in 5-mL sealed vials at 180°C for 20 min, and sampled every 5
min. Before analysis, vials were cooled and stored at room temperature for 24 h. A SPME
fiber (DVB/CAR/PDMS) was used for sampling volatile compounds. Vials were conditioned at
40°C for 5 min and the volatile compounds were adsorbed for 5 min at 40°C. The SPME fiber
was desorbed at 240°C for 3 min in the GC/MS injector. A capillary GC column Rtx-Wax (30 m
x 0.25 mm i.d. x 0.25 m f.t.) coated with polyethylene glycol was used. The temperature
was programmed from 45°C to 200°C at 3°C/min. The injector and the ion source
temperatures were set at 240°C and 200°C, respectively. Helium was used as carrier gas at
36.2 cm/s. The injection was performed in the split mode (1:30). The electron energy was 70
eV. A mass range from 40 to 250 m/z was scanned at a rate of 1250 amu/s. Some
compounds that derived from the decomposition of side-chain peroxides (2-heptanone, 6methyl-2-heptanone, 3-methylpentanone and 3-methylbutanoic acid), were selected as
markers of cholesterol oxidation. The suitability of the method was also tested on egg yolk
powder and milk powder. The analytical performance of the SPME-GC/MS method together
with the consequent significant reduction of the analysis time and consumables,
demonstrates its great potential for rapid screening of cholesterol oxidation.
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3rd ENOR symposium – Swansea 2013
Isotopically Labelled Girard Reagents for Multiplexed Analysis of Oxysterols
and Cholestenoic Acids in Plasma and CSF
Peter J. Crick, T. William Bentley, Ian Matthews, Yuqin Wang
and William J Griffiths
Institute of Mass Spectrometry, College of Medicine, Swansea University
The analysis of oxysterols and cholestenoic acids in biological matrices is complicated by
their low abundance (pg/mL to ng/mL), especially when compared to cholesterol (µg/mL to
mg/mL), lack of chromaphore, and poor ionisation profile in mass spectrometry. Some of
these problems can be overcome by chemical derivatisation prior to analysis by either
GC/MS or, more recently, LC/MS.
Our group has developed an analytical strategy based on oxidation with cholesterol oxidase
followed by derivatisation with the Girard P reagent. We have termed this EADSA (enzymeassisted derivatisation for sterol analysis). The Girard P derivative incorporates a
permanently charged quaternary ammonium ion leading to greatly enhanced sensitivity in
ESI-MS. Additionally, predictable fragmentation yields structurally informative MS n spectra
that allow identification of up to 40 metabolites.
We have now expanded this method by incorporating isotope labels into the Girard P
reagent. This allows the analysis of up to six samples in a single LC/MS run. As well as making
more efficient use of instrument time this improves accuracy of quantitation by allowing a
direct comparison of the analytes of interest. We have applied this method to plasma and
CSF samples both with and without prior hydrolysis of sterol esters.
Here, we illustrate our approach with a rapid identification and detailed analysis of plasma
from patients with Smith-Lemli-Opitz syndrome (SLOS) caused by a deficiency in 7dehydrocholesterol reductase.
We also present an optimised procedure for the analysis of oxysterols in as little as 30 µL CSF
allowing the identification and quantitation of over twenty oxysterols and cholestenoic
acids.
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3rd ENOR symposium – Swansea 2013
On the formation of 7-ketocholesterol
Ulf Diczfalusy, Anita Lövgren Sandblom and Ingemar Björkhem
Division of Clinical Chemistry, Department of Laboratory Medicine, Karolinska Institutet,
Stockholm, Sweden
7-Ketocholesterol is a major cholesterol oxidation product formed e.g. during in vitro
oxidation of low density lipoprotein. It accumulates in human atherosclerotic plaques and
displays a multitude of biological effects in in vitro experiments. A new mechanism for the
formation of 7-ketocholesterol was recently described involving enzymatic conversion of 7dehydrocholesterol to 7-hydroxylase
1
(CYP7A1) with concomitant formation of cholesterol-7,8-epoxide .
Patients with the rare inborn disorder Cerebrotendinous Xanthomatosis (CTX) have elevated
levels of 7-dehydrocholesterol in the circulation and a strongly upregulated CYP7A1. We
hypothesized that these patients may have a production of 7-ketocholesterol from 7dehydrocholesterol by the proposed new mechanism. Analysis of plasma from a CTX patient
revealed elevated levels of 7-ketocholesterol and 7-dehydrocholesterol as well as a
significant level of cholesterol-7,8-epoxide, which is not found in plasma of healthy
volunteers.
Treatment of this patient with chenodeoxycholic acid resulted in
downregulation of CYP7A1 and was accompanied by significantly decreased levels of 7ketocholesterol, 7-dehydrocholesterol and cholesterol-7,8-epoxide in plasma. These results
suggest that 7-ketocholesterol may be formed enzymatically from 7-dehydrocholesterol in
vivo.
1Shinkyo,
R., Xu, L., Tallman, K. A., Cheng, Q., Porter, N. A., and Guengerich, F. P. (2011)
Conversion of 7-dehydrocholesterol to 7-ketocholesterol is catalyzed by human cytochrome
P450 7A1 and occurs by direct oxidation without an epoxide intermediate. J. Biol. Chem. 286,
33021-33028 .
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3rd ENOR symposium – Swansea 2013
First worldwide non-cholesterol and oxysterol survey
Dieter Lütjohann
Institute for Clinical Chemistry and Clinical Pharmacology, University of Bonn
Non-cholesterol sterols (NCS), which encompass endogenous cholesterol precursors
(lanosterol, lathosterol, desmosterol) and exogenous phytosterols (campesterol, sitosterol,
5-campestanol, 5-sitostanol) as well as cholesterol metabolites (5-cholestanol; bile acid
precursors/oxysterols: 7-, 24S-, and 27-hydroxycholesterol; other oxysterols: 7- and 25hydroxycholesterol, 7-ketocholesterol), are widely used in biomedical research as surrogate
markers for estimating cholesterol synthesis, cholesterol absorption efficiency, bile acid
synthesis and radical oxygen species (ROS)-status. Furthermore, NCS and oxysterols are used
as prognostic and diagnostic tools for the description of dyslipidemic and neurodegenerative
states in mammalian subjects. These NCS and oxysterols show even stronger correlation
with cholesterol absorption or synthesis or bile acid synthesis when expressed as ratios to
total cholesterol, which standardizes for variations in sterol transport protein
concentrations. Specifically, when reporting NCS in a ratio to cholesterol, the cholesterol
measurement should ideally be from the same sample preparation as the NCS, and
measured by the same chromatographic method.
Twenty laboratories specialized in chromatographic lipid analysis, either gas or liquid
chromatography, agreed to participate in a first worldwide survey under the expertise of the
reference institute for bioanalytics (RfB) located at Bonn, Germany. A set of two different
lyophilized pool sera (A and B) was sent to each participant and the results were sent back
from 18 participants and evaluated by RfB. The different sterols, stanols and oxysterols were
quantified either by GC-flame-ionization detection, GC-mass selective detection or LC-mass
selective detection. Additionally, different internal standards were used: 5a-cholestane
(mostly for GC-FID and GC-MS of cholesterol or NCS), epicoprostanol (for NCS), deuterium
labeled sterols/oxysterols (isotope dilution mass spectrometry, GC- and LC-MS) and in one
case 19-OH-cholesterol for oxysterols. The data were given in the individual units used by
the participant and converted into standard units for comparison (cholesterol and NCS as
mg/dL; oxysterols as ng/mL). Each participant received a clear overview of his position in
form of Youden-Plots and basic statistical evaluation (mean, median, SD, min, max).
Unfortunately, cholesterol was autoxidized as proofed by extremely high concentrations of
7- and 7-hydroxycholesterol as well as by increased 7-ketocholesterol. Those oxysterols,
which are exclusively enzymatically produced (24S-, 25- and 27-hydroxycholesterol) were in
the range as known from the literature and are integrated in the evaluation process.
The lowest coefficient of variation is presented for cholesterol (A: 25.1% and B: 21.5%). For
non-cholesterol sterols there are coefficients of variation between 54% and 189%. For
oxysterols, except 7-oxygenated cholesterol, coefficients of variation vary between 36% and
125%.
Conclusion: We need further careful considerations and proposals to harmonize the
methods for GC- and LC-analytics of cholesterol, non-cholesterol sterols/stanols and
oxysterols. This includes the work-up procedure, safety parameters, use of standards and
internal standards, chromatographic separation and detection mode and finally the unit to
present each parameter.
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3rd ENOR symposium – Swansea 2013
Macrophages, viruses and Ch25h
Peter Ghazal
University of Edinburgh
The sterol metabolic network is emerging as an intrinsic participant of the immune response.
Recent studies have begun to elucidate the molecular mechanisms linking interferon with
the sterol network and the identity of sterol mediators in this process. In particular, evidence
for a cellular antiviral role for macrophage production of 25-hydroxycholesterol (cholest-5en-3β,25-diol, 25HC) as a component of the sterol metabolic network linked to the innate
immune response via Stat1 has been documented. New data suggests a further role in
mediating silencing of infection in latently infected macrophages, as part of a novel host
directed feed-forward repression mechanism. This talk will discuss these emerging
physiological and mechanistic principles underpinning the role of sterol metabolism in
antiviral immunity.
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3rd ENOR symposium – Swansea 2013
Evaluation of the therapeutic potential of LXR antagonists for X-linked
adrenoleukodystrophy
Catherine Gondcaille, Emmanuelle C. Genin, Tatiana E. Lopez,
Alexandre M.M. Dias, Flore Geillon, Pierre Andreoletti,
Mustapha Cherkaoui-Malki, Thomas Nury,
Gérard Lizard, Doriane Trompier and Stéphane Savary
Laboratoire Bio-PeroxIL, EA7270 University of Burgundy, 6 Bd Gabriel,Dijon, F-21000, France
X-linked adrenoleukodystrophy (X-ALD, OMIM 300100) is the most frequent peroxisomal
disorder with an incidence of 1/17 000 births. The biochemical hallmark of this
neurodegenerative disease is the accumulation of very-long-chain fatty acids (VLCFA, fatty
acids with a carbon chain longer than 22 carbons) in plasma and tissues resulting from a oxidation defect. Oxidative stress and inflammation are also key components of the
pathogenesis of X-ALD. X-ALD is caused by mutations in the ABCD1 gene, which encodes for
a peroxisomal half ABC transporter predicted to participate in the entry of VLCFA-CoA into
the peroxisome, the unique site of their -oxidation. Two homologous peroxisomal ABC
transporters, ABCD2 and ABCD3 have been proven to compensate for ABCD1 deficiency
when overexpressed. Pharmacological induction of these target genes could therefore
represent an alternative therapy for X-ALD.
Since LXR activation was shown to repress ABCD2 expression, we investigated the effects of
LXR antagonists in murine 158N oligodendrocytes, human HepG2 hepatoma cells and human
skin fibroblasts from X-ALD patients. Cells were treated for 3 days with GSK (17) (a LXR
antagonist recently discovered from the GlaxoSmithKline compound collection), 22(S)hydroxycholesterol (22S-HC, another LXR antagonist) and 22R-HC (an endogenous LXR
agonist). Due to the toxic effect of LXR ligands, results obtained with murine 158N cells were
unconclusive. However, we observed induced gene expression of ABCD2, ABCD3 and
CTNNB1 (the gene encoding for -Catenin) in HepG2 cells and in X-ALD fibroblasts treated
with LXR antagonists. Interestingly, such induction in X-ALD fibroblasts was concomitant with
a decrease of oxidative stress.
These results open an additional field of investigation for LXR antagonists as therapeutic
agents in the context of demyelinating neurodegenerative disorders.
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3rd ENOR symposium – Swansea 2013
Argan oil sterols as potential new LXR agonists
Youssef El Kharrassi1,2, Mohammad Samadi3, Tatiana Lopez1,4, Riad El Kebbaj1,2,
Pierre Andreoletti1, Khadija Moustaid2, Hammam I. El Hajj1, Norbert Latruffe1,
Joseph Vamecq5, M’Hammed Saïd El Kebbaj6, David Masson3,
Stéphane Savary1, Gérard Lizard1, Boubker Nasser2
and Mustapha Cherkaoui-Malki1.
1Laboratoire
Bio-PeroxIL, EA7270, Université de Bourgogne, Dijon, France;
de Biochimie et Neurosciences, FST Settat, Université Hassan I, Morocco;
3LCPMC-A2, ICPM, Department of Chemistry, Université de Lorraine, Metz, France;
4CRINSERM 866, Dijon, France;
5INSERM and HMNO, CBP, CHRU Lille, France;
6LRLA, Faculté des Sciences Ben M’sik, Université Hassan II-Mohammedia-Casablanca,
Morocco.
2Laboratoire
Argan edible oil is obtained by cold-pressure of roasted kernels from Argania spinosa [L.]
Skeels, a singular Mediterranean species developing in the southwestern region of Morocco.
Argan oil is used as a traditional food ingredient in the ‘amazigh diet’, bringing almost 25% of
total diet fat intake to indigenous consumers. Health benefits of this delectable virgin oil are
highlighted by several studies documenting its cardiovascular protective potential including
hypocholesterolemic and hypotriglyceridemic properties in consumer populations. Lowering
of plasma LDL-cholesterol and lipoperoxides concomitant to a higher plasmatic tochopherol
concentration was rapidly pointed out by early clinical studies on argan oil, underlying
mediators of these health benefits still remaining to be clarified.
In this context, our main objective was to puzzle out the role of each constitutive ingredient
in the protective effects of argan oil. Because several sterol derivatives have been shown to
master regulators of several metabolic pathways, including lipid and cholesterol metabolism
as well as inflammation and innate immunity, our studies focused on argan oil sterols. For
this purpose, the chemical syntheses with subsequent purifications of two major argan
sterols (schottenol and spinasterol) commercially unavailable were performed. In the brain,
LXR agonists have been shown to regulate lipoproteins synthesis and cholesterol efflux in
astrocytes and in microglia. The latter also participates to brain inflammatory processes.
Furthermore, LXR activation has been shown to have significant anti-inflammatory
properties. After testing their cytotoxic effects on the murine microglia BV2 cells (MTT test),
these cells were exposed to each argan oil sterol. Expression of LXR-target genes involved in
cholesterol efflux, ABCA1 and ABCG1, was determined using a real-time PCR. Activation of
LXR using the gene reporter system based on LXRE-activating luciferase gene expression was
also studied. Results of these preliminary molecular investigations will be discussed as
regards to the role(s) of LXR in cholesterol metabolism in microglial cells.
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3rd ENOR symposium – Swansea 2013
Impairment of cholesterol metabolism in Huntington Diseases: evidence of
anabolic dysfunction in neurodegenerative process
Valerio Leoni and Claudio Caccia
Laboratory of Clinical Pathology and Medical Genetics, Foundation IRCCS Institute of
Neurology Carlo Besta, via Celoria 11, 20133 Milano (MI), Italy
Huntington’s Disease (HD) is an autosomal dominant neurodegenerative disorder caused by
an abnormal expansion of CAG trinucleotide repeat in the Huntingtin (HTT) gene. A gradual
atrophy of the striatum and astrogliosis result into a severe cortical atrophy combined with
striatal degeneration at MRI.
The expression of some genes involved in the cholesterol biosynthetic pathway as HMGCoA
reductase, Cyp51 and DHCR7, the brain amounts of the cholesterol, lanosterol and
lathosterol together with 24OHC were found reduced in brain, striatum and cortex in HD
mice models and cell lines.
mtHTT decreases the amount of active SREBP and cannot up-regulate LXR and LXR-targeted
genes (SREBP, Abcg1 and Abcg4, HMGCoA reductase), with lower synthesis and transport of
cholesterol from astrocytes to neurons. In primary oligodendrocytes, mtHTT was found to
inhibit the regulatory effect of PGC1α on cholesterol metabolism and the expression of MBP.
In pre-manifest individuals 24OHC decreased progressively getting closer to the clinical onset
proportionally to cognitive and neuropsychiatric dysfunction and MRI brain atrophy. A
significant reduction of cholesterol levels were however found only in advanced stages but
lanosterol and lathosterol, together with 27OHC and 24OHC were reduced in plasma
collected from HD patients at any disease stage.
Abnormalities in mitochondrial dynamics and functions, dysfunction in intermediary
metabolism (as procatabolic syndrome and weight loss) were described in HD patients.
We observed abnormalities in cholesterol and fatty acids in samples from HD mice.
We also observed alteration of the plasma acyl-carnitines profile suggestive of beta
oxidation dysfunction in HD patients.
Huntingtin seems to play a regulatory role in lipid metabolism. Its mutation was found
associated to an impairment of cholesterol metabolism proportional to the CAG repeat
length, the mtHtt load and the disease burden in patients. The reduced synthesis of AcetlyCoA and dysfunction of mitochondrial OXPHOS can contribute to the modification of
cholesterol and fatty acids profile observed in models and patients. To the neurotoxic effect
of mtHtt may be associated an anabolic dysfunction of lipid metabolism.
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3rd ENOR symposium – Swansea 2013
Oxysterols, Liver X Receptors and Male Reproduction
Salwan Maqdasy
1) Génétique Reproduction et Développement ; Team "Lipids, Nuclear Receptors and Male
Disorders" ; CNRS 6293 INSERM U1103 Clermont Université
2) Service d’endocrinologie, diabétologie, maladies métabolique, CHU Clermont-Ferrand,
Clermont Université
Oxysterol nuclear receptors LXRα and LXRβ regulate intracellular cholesterol levels. LXRs are
expressed in different cellular compartments in the testis; phenotypic analysis of mice
lacking genes coding LXRs (lxrαβ-/-) enlighten their physiological role in testicular function.
These mice are prematurely sterile and characterized by structural and functional
alterations. In order to investigate in vivo functions of LXRβ, we generated rescued lxrαβ-/mice with LXRβ re expression in Sertoli cells (lxrαβ-/-Tglxrβ). The re expression was
undertaken by additive transgenesis of cDNA coding this receptor under control of Amh
promoter.
LXRβ restores lipid homeostasis in Sertoli cells. It participates in the regulation of most of
Sertoli cell functions. Furthermore, proliferation of germ cells is controlled by LXRβ
expressed in Sertoli cells. This receptor plays a paracrine role in regulating Star and 3βhsd
transcription, genes coding enzymes of steroidogenesis. Nevertheless, persistent sterility in
these mice demonstrates that LXR expression in other cellular compartments, especially in
germ cells, play an important role in the maintenance of fertility.
Key words: LXR, Oxysterols, Sertoli cell, testis, hypo fertility, lipid homeostasis
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3rd ENOR symposium – Swansea 2013
GC-QToF with atmospheric chemical ionization - a new approach to detect
oxysterols
S. Matysik1, F.M. Matysik2, S. Bauer2, J. Kiermaier2 and G. Schmitz1
1 University
Hospital Regensburg, Institute of Clinical Chemistry and Laboratory Medicine,
Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany
2 University of Regensburg, Institute of Analytical Chemistry, Chemo- and Biosensors, 93040
Regensburg, Germany
We describe the optimization and application of an atmospheric pressure chemical
ionization (APCI) interface to couple gas chromatography (GC) to time-of-flight (ToF) mass
spectrometry (MS). Although several configurations of GC-APCI-MS systems have been
described this technique has not been adopted broadly and it is not typically used in clinical
laboratories. The analytical performance of GC-APCI-MS systems depends on several details
of the APCI source and the corresponding parameter setting. To explore the potential of GCAPCI-MS the APCI source conditions were optimized using several standard compounds,
namely benzophenone, methyl palmitate, methyl stearate, and cocaine hydrochloride.
Nitrogen with variable water content was introduced to the source to provide a well-defined
concentration of water molecules for chemical ionization. Therefore, the APCI source was
modified by implementation of a sensor for humidity to monitor the water content inside
the source. In addition, other source parameters including drying gas flow rate and
temperature, make-up gas pressure, corona voltage were studied. Three repeated injections
of the test mixture were carried out for each parameter setting in order to assess the
repeatability of the response.
The optimized source parameters were used to perform oxysterol determinations in human
plasma after liquid/liquid extraction and trimethylsilyl derivatization. In particular, 24-, 25-,
27-hydroxycholesterol, 7-hydroxycholesterol and 7-ketocholesterol were determined in
fullscan mode taking advantage of the high mass accuracy of ToF-MS detection. Calibration
plots were linear with R2 better than 0.99 in concentration ranges between 10 and 300
ng/ml. The high mass accuracy with m/z in the range of 1 ppm enabled screening for 7hydroxy-4-cholesten-3-one, several phytosterols and cholesterol precursors.
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3rd ENOR symposium – Swansea 2013
Cholesterol aldehydes: The mechanisms of their formation and pathological
roles in oxidative stress related-disorders
Noriyuki Miyoshi, Susumu Tomono and Hiroshi Ohshima
University of Shizuoka, Shizuoka, Japan
Cholesterol aldehydes, 3β-hydroxy-5-oxo-5,6-secocholestan-6-al (secosterol-A), and its
aldolization product, 3β-hydroxy-5β-hydroxy-B-norcholestane-6β-carboxaldehyde (secosterol-B),
are major products formed by cholesterol ozonolysis. Elevated levels of secosterol-A and -B
have been detected in human atherosclerotic plaques and brain tissues from patients with
neurodegenerative diseases such as Alzheimer’s disease and Lewy body dementia.
Secosterols can modify covalently proteins, including apolipoprotein CII, amyloid-β peptide,
α-synuclein, and p53, which accelerate amyloidogenesis and fibrilization of these proteins.
Thus secosterols have been postulated to play pivotal roles in the pathogenesis of
atherosclerosis and neurodegenerative diseases. We have previously developed a highly
sensitive method for analysis of secosterols and their derivatives using LC-ESI-MS/MS. Using
the method, we have shown that secosterols could be formed in vivo via cholesterol
oxidation with reactive oxygen species generated in myeloperoxidase-dependent and
independent pathways. Moreover we have reported for the first time that ozonolysis
products of cholesteryl oleate and linoleate, 9-oxononanoyl-secosterols, were present at low
concentrations in human LDL specimens isolated from healthy subjects. These secosterols
and their derivatives were also found to be strongly cytotoxic, induce protein aggregation
and inhibit neuronal and endothelial nitric oxide synthases. We will discuss pathological
roles of secosterols in several oxidative stress associated-diseases.
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3rd ENOR symposium – Swansea 2013
Diverse functions of 24(S)-hydroxycholesterol in the brain
Noriko Noguchi, Yoshiro Saito and Yasuomi Urano
24(S)-hydroxycholesterol (24S-OHC) which is enzymatically produced in the brain plays
important physiological roles in maintaining the brain cholesterol homeostasis. We have
shown that 24S-OHC down-regulates amyloid precursor protein (APP) trafficking via
enhancement of the complex formation of APP with up-regulated glucose-regulated protein
78, an ER chaperone. By this mechanism, 24S-OHC suppresses amyloid- (A) production in
human neuroblastoma SH-SY5Y cell. Furthermore, 24SOHC at sub-lethal concentrations
induces adaptive response via transcriptional activation of liver X receptor signaling
pathway, thereby protecting neuronal cells against the forthcoming oxidative stress induced
by 7-ketocholesterol. These results may support data shown in mouse models of Alzheimer’s
disease (AD) in which increasing 24S-OHC levels reduced AD pathology. On the other hand,
the increase of 24S-OHC in plasma and cerebrospinal fluid of AD patients and cytotoxicity of
24S-OHC have been reported. W have shown that high concentrations of 24S-OHC induce
neuronal cell death by necroptosis, a form of programmed necrosis. This caspaseindependent cell death by 24S-OHC is not inhibited by antioxidants. In this article, we show
the diverse functions of 24S-OHC and indicate importance of control of 24S-OHC levels in the
brain for preventing neurodegenerative diseases.
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3rd ENOR symposium – Swansea 2013
Induction of a dual mode of cell death (autophagy and apoptosis) on
7-ketocholesterol-treated 158N murine oligodendrocytes
Thomas Nury 1, Margaux Doria 1, Amira Zarrouk 1,2, Hervé Begue 1,
Anne Vejux 1, Mohammad Samadi 3, Régis Delage-Mourroux 4
and Gérard Lizard 1
1
Laboratoire Bio-PeroxIL, EA7270, Université de Bourgogne / INSERM, Dijon, France; 2
Laboratoire de Biochimie, LR12ES05, LR-NAFS ‘Nutrition - Functional Food & Vascular Health’,
Faculté de Medicine - Université de Monastir, Tunisia ; 3 LCPMC-A2, ICPM, Department of
Chemistry, Université de Lorraine, Metz, France ; 4 Université de Franche-Comté, UFR Sciences
et Techniques, EA 3922/IFR133, Besançon, France.
Apoptosis and autophagy are crucial mechanisms regulating cell death, and the relationships
between apoptosis and autophagy are still not well known. It was previously reported on
human monocytic U937 cells that 7-ketocholesterol (7KC) induces a complex mode of cell
death associated with apoptotic characteristics, enhanced superoxide anion production, and
probably also autophagic characteristics (presence of monodansylcadaverine-positive
cytoplasmic structures and multilamellar cytoplasmic structures). It was therefore suggested
that 7KC-induced cell death could constitute a particular entity for which the term
oxiapoptophagy (OXIdation, APOPTOsis, and autoPHAGY) was proposed (Monier S et al. ,
Biochem Biophys Res Commun 2003; 303(3): 814-824).
On murine oligodendrocytes 158N, 7KC (20 µg/mL, 24 h) induced a complex mode of cell
death characterized by a loss of transmembrane mitochondrial potential, enhanced
cytoplamic membrane permeability to propidium iodide, lysosomal destabilisation, caspases
activation (especially caspase-3), internucleosomal DNA fragmentation, and condensation
and/or fragmentation of the nuclei. These different criteria are characteristic of an apoptotic
mode of cell death. Moreover, exposure of 158N cells to 7KC promotes conversion of
microtubule-associated protein light chain 3 (LC3-I) to LC3-II which is a characteristic feature
of autophagy. Modulations of p62 expression was also observed under treatment with 7KC.
Noteworthy, 7KC-induced cell death was prevented, and apoptosis and autophagy were
suppressed by treating the cells with VitE (alpha-tocopherol, 400 µM). In the presence of the
wide spectrum caspase inhibitor (z-VAD-fmk, 100 µM), 7KC-induced apoptosis was
prevented and autophagy was strongly reduced.
Altogether, our data provide evidences that 7KC simultaneously induces autophagy and
apoptosis on 158N cells. This model could constitute a useful tool to precise the
relationships between autophagy and apoptosis as well as to identify cellular targets and
drugs (as efficient as VitE) allowing to inhibit the dual mode of cell death induced by 7KC.
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3rd ENOR symposium – Swansea 2013
Oxysterol-binding proteins: Sterol and phosphoinositide sensors with
versatile functions in cell regulation
Vesa M. Olkkonen, Henriikka Kentala, You Zhou and Marion Weber-Boyvat
Minerva Foundation Institute for Medical Research, Biomedicum 2U, FI-00290 Helsinki,
Finland
Cytoplasmic oxysterol-binding protein (OSBP)-related proteins (ORPs) are lipid
binding/transfer proteins implicated in a variety of cellular functions: in lipid
metabolism/transport, vesicle transport and signaling cascades. They possess a characteristic
sterol-binding domain, which accommodates oxysterols and/or cholesterol. In mammals a
large number of ORPs are encoded by 12 genes. Ten members of the OSBP/ORP family are
anchored to ER membranes either via interactions with type 2 integral membrane proteins
called VAMP-associated proteins (VAPs) or via a trans-membrane segment. In addition, most
ORPs carry N-terminal plecktrin homology (PH) domains that bind phosphoinositides and
target non-ER membranes. Several ORPs are known to localize and function at membrane
contacts sites (MCS) at which ER is closely apposed with other organelles. Such sites have
well documented roles in lipid syntheses/transport, calcium fluxes, and signaling processes.
We investigated systematically the spatial occurrence and regulation of the interaction of
OSBP/ORPs with VAP proteins by employing the Bimolecular Fluorescence Complementation
(BiFC) technique. The interactions were in several cases spatially restricted, localizing to
distinct sites that most likely represent MCSs, an interpretation supported by electron
microscopic observations. Analysis of sterol-binding deficient ORP mutants suggested that
ORP sterol liganding negatively regulates ORP-VAP interactions and thereby MCS formation
or function. ORP1L-VAP complexes encircled Rab7-positive late endosomes (LE), consistent
with the documented role of ORP1L in mediating ER-LE contacts. ORP2-VAP complexes
localized to ER domains with closely associated lipid droplets, and were shown to inhibit the
hydrolysis of cellular TAGs. Consistently, ORP2 and VAP silencing by RNA interference
resulted in a dysregulation of TAG metabolism. The ORP2 ligand 22(R)hydroxycholesterol
was shown to reduce binding of ORP2 to VAP and to detach LD from the ER. Association of
ORP3 with VAPs was shown to be regulated by phosphorylation of the ORP, which induced
selective binding of the hyper-P form to VAP. ORP3-VAP-complexes were, in addition to
reticular ER, seen associated with patches of the plasma membrane (PM), putative ER-PM
MCSs, where they may execute a signaling function.
The present work, together with other recent studies, suggests a complex role of ORPs as
sterol/phosphoinositide sensors modifying MCS function in lipid metabolism and signaling.
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3rd ENOR symposium – Swansea 2013
Discovery of Dendrogenin A as a product of cholesterol and histamine
metabolism with cell differentiation and anti-tumour properties.
Sandrine Silvente-Poirot1,2,3 and Marc Poirot1,2,3
1INSERM
UMR 1037, Team « Sterol Metabolism and Therapeutic Innovations in Oncology»,
Cancer Research Center of Toulouse, Toulouse, France;
2University Toulouse III, Toulouse France,
3Institut Claudius Regaud, Toulouse, France;
We previously synthesized dendrogenin A (DDA) based on the hypothesis that it could be a
natural metabolite occurring in mammals through the reaction of the 5,6-epoxide of
cholesterol with histamine that can concentrate at the level of the microsomal anti-estrogen
binding site (AEBS), as it can bind both molecules (de Medina et al, J Med Chem, 2009). The
AEBS contains Cholesterol-5,6-Epoxide Hydrolase (ChEH) and is the target for a number of
drugs and natural compounds that are known to induce tumour cell differentiation and
death (Silvente-Poirot S & Poirot M, Curr Opin Pharmacol, 2012). We explored this
hypothesis and reported the discovery of DDA in mammalian tissues and normal cells as an
enzymatic product of the conjugation of 5,6-epoxycholesterol and histamine (de Medina et
al, Nat Commun, 2013).
Interestingly, DDA was not detected in cancer cell lines and was 5-fold lower in human
breast tumours compared with normal tissues, suggesting a deregulation of DDA metabolism
during carcinogenesis. We established that low concentrations of DDA selectively inhibited
the Cholesterol-5,6-Epoxide Hydrolase (ChEH) which is carried out by the AEBS and triggered
differentiation and growth control in vitro and in vivo in syngeneic tumours implanted into
mice, and improved animal survival. The properties of DDA and its decreased level in
tumours suggest a physiological function in maintaining cell integrity and differentiation.
Here we explored the anti-tumour effects of high concentrations of DDA and its mechanism
of action. We found that higher concentrations of DDA induced active cell death in human
and murine tumour cells of different origins in vitro and in vivo. We determined that the cell
death induced by DDA was mainly of an autophagic nature and involved the stimulation of
the expression of two transcription factors NR4A1 (Nur77, NGFIB) and NR4A3 (Nor1). Finally,
we established that DDA was a ligand and a selective modulator of the liver-X-receptor (LXR)
and that LXRcontrolled the induction of the cytotoxic autophagy by DDA in tumour cells.
These data established that DDA is a new LXR ligand that controls cytotoxic autophagy.
In conclusion, the discovery of DDA reveals a new metabolic pathway at the crossroads of
cholesterol and histamine metabolism and the existence of a new class of amino-oxysterols
that control ChEH activity and are modulators of LXR. Overall our data indicate that DDA is
an endogenous cholesterol derivative that may help to maintain cell integrity through cell
differentiation and autophagic cell death.
More generally, our data highlight a positive effect of a cholesterol metabolite against the
occurrence and the development of cancer.
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3rd ENOR symposium – Swansea 2013
Tandem mass tags for targeted steroidomics using a shot-gun ESI-MS method
K. Rigdova1, W. J. Griffiths1, Y. Wang1 and M. Ward2
1Institute
of Mass Spectrometry, College of Medicine, Swansea University, Swansea, UK.
Sciences plc, Surrey, UK.
2Proteome
A novel concept for steroid identification utilizing tandem mass tags (TMTs) as derivatization
reagents has been developed exploiting shotgun electrospray ionization-mass spectrometry
(ESI-MS) and targeting analysis on oxosteroids.
The presented derivatization reagents are isotopically labelled tandem mass tags, specifically
designed to allow the relative and absolute quantitation of derivatized steroids (“light” v
“heavy” tags) in simple MS spectra. As an alternative to isotopic tags, a panel of isobaric
tags, with no difference in nominal mass, but yielding different mass reporter ions in tandem
mass (MS/MS) spectra has been exploited. When using this multiplexed approach,
simultaneous analysis of a set of samples can be achieved, increasing the throughput of
quantitative steroidomic assays.
The derivatization reaction was applied and optimized to 20 steroid standards. TMT tags
were tested on a urine sample of adrenal cortical carcinoma (ACC) origin, revealing
differences in relative abundance of steroid metabolites when compared to control urine
samples.
The use of isobaric 6-plex tags for quantification was exploited on selected urine metabolites
in a ACC sample, demonstrating the practicality of the developed approach.
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3rd ENOR symposium – Swansea 2013
Quantification of oxysterols in β-catenin deficient pancreatic cancer cells
using nano-AFFL-SPE-LC-ESI-MS/MS
Hanne Røberg-Larsen1, Nina T. Solberg2, Petter A. Olsen2, Kaja Lund2,
Martin F. Strand2, Stefan Krauss2 and Steven R. Wilsen1
1Department
2Unit
of Chemistry, University of Oslo
for cell signaling, SFI-CAST innovation Center, Oslo University hospital
In cancer drug development cell signaling pathways such as the hedgehog and Wnt pathway
is of great interest. However, to understand how drug candidates work, the signaling
pathways must be studied. Oxysterols can activate the hedgehog pathway, but their main
role is still unknown.
Limited sample sizes and low concentration challenge the analytical techniques available.
Using nano-SPE-LC provides better detection limits, however nano-SPE-LC methods are not
known as robust. We have therefore developed and validated an automatic filtration and
filter back-flush (AFFL) system for nano-SPE-LC for determination of oxysterols in small
samples sizes. With this system, oxysterols can be detected from <10 000 cells. Oxysterols
were “charge-tagged” with Girard T reagent prior analysis and only one sample transfer step
was required for the entire process of cell lysis, derivatization and determination of selected
oxysterols. Quantification limits for 24S-, 25- and 27-hydroxycholesterol was 13 pM (~0.065
fmol /~33 fg injected on column).
The method is used on β-catenin deficient pancreatic cancer cells to study the role of
oxysterols in the hedgehog and Wnt signaling pathways.
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3rd ENOR symposium – Swansea 2013
A microRNA targeting SREBP2 and coupling the antiviral interferon response
to the regulation of cholesterol biosynthesis
Kevin A. Robertson and Peter Ghazal
Division of Pathway Medicine, University of Edinburgh
Recent studies have shown that the sterol metabolic network has a central role in immunity
and inflammation and functions as an intrinsic component of the interferon-elicited antiviral
response. How interferon (IFN) regulates the sterol network is poorly understood. Using a
high-resolution, systematic, temporal analysis of RNA synthesis and abundance we have
demonstrated a reduction in cholesterol biosynthesis-related transcript synthesis and
abundance in IFN-gamma-treated macrophages. These decreases are co-incident with
increased expression of a miRNA whose up-regulation is interferon-dependent in the context
of viral infection. We show that the miRNA specifically targets the SREBF2 transcript and can
decrease SREBP2 protein abundance. Importantly, it can also regulate intracellular
cholesterol concentration and inhibits the replication of human and mouse herpesviruses
and Influenza A virus (H1N1). These findings provide the first evidence for a host, interferonregulated miRNA that modulates cholesterol biosynthesis and is anti-viral
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3rd ENOR symposium – Swansea 2013
7-ketocholesterol as marker of cholesterol oxidation in model and food
systems: when and how
Maria Teresa Rodriguez-Estrada1 and Guadalupe Garcia-Llatas2
1
Department of Agricultural and Food Sciences, Alma Mater Studiorum-University of Bologna
(Italy); Nutrition and Food Chemistry Area, Faculty of Pharmacy, University of ValenciaBurjassot (Spain)
Cholesterol can oxidize by either enzymatic or chemical mechanisms, generating a wide
range of oxidation products (COPs) with adverse biological effects. COPs are characterized
by different functional groups and are produced in different ratios/amounts depending on the
treatment and storage conditions. To follow the cholesterol oxidation process, 7ketocholesterol (7-k) has been often used as marker in both model and food systems as it is
easily formed and it is one of the most representatives ring COPs. However, 7-k does not
always rise with increasing time/temperature conditions, especially in complex systems, highprotein or highly stressed food. The following review provides a critical picture of the
utilization of 7-k as marker in model and food systems, focusing on the possible causes and
effects of such different behaviors and trends, as well as on the advantages and
disadvantages of this choice when the extent of cholesterol oxidation is to be depicted.
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3rd ENOR symposium – Swansea 2013
Signaling of the oxysterol / EBI2 pathway in immune cells
Inga Preuss, Hans Voshol, Yann Abraham, Marie-Christine Lasbennes,
Stéphane Laurent, Nina Baur, Birgit Baumgarten, Juan Zhang, Francois Gessier,
Klaus Seuwen and Andreas W. Sailer
Novartis Institutes for BioMedical Research, Basel, Switzerland
7, 25-dihydroxycholesterol (7, 25-OHC) and closely related oxysterols have recently been
identified as potent and selective activators of a G protein-coupled receptor called EBI21,2
(Epstein-Barr virus induced gene 2, aka GPR183). The EBI2 receptor is required for humoral
immune responses and has been linked to autoimmune disease. Similarly mice deficient in
cholesterol 25-hydroxylase (Ch25h)1, the key enzyme for production of 7, 25-OHC, fail to
position activated B cells within the spleen to the outer follicle and mount a reduced plasma
cell response after an immune challenge.
Activation of the EBI2 receptor shapes the immune response by directing cell migration of
EBI2-expressing cells to the site of ligand production. To investigate the cellular signaling
events that occur in that process, we have used the monocyte cell line U937 to perform
quantitative phospho-proteomics using stable isotope labeling with amino acids in cell
culture (SILAC) and mass spectrometry. In order to explore the specificity of oxysterolstimulated protein phosphorylation, we also conducted experiments using SDF1, a cytokine
activating the CXCR4 receptor. Phosphoproteins specific to oxysterol activation as well as
common to both chemoattractants are being characterized.
Quantitative phosphoproteomics has emerged as a powerful tool to study cell signaling on a
proteome-wide scale. A detailed understanding of the biochemical mechanism of receptor
signaling and trafficking will help to delineate the role of the oxysterol/EBI2 system in health
and disease.
1 Hannedouche
et al. Nature 475(2011)524 / 2 Liu et al. Nature 475(2011)519
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3rd ENOR symposium – Swansea 2013
7-hydroxycholestrol as a possible biomarker of cellular lipid peroxidationdifference between cellular and plasma lipid peroxidation
Yoshiro Saito and Noriko Noguchi
Faculty of Medical and Life Sciences, Doshisha University
Our research group has been developed a method for the measurement of lipid peroxidation
in vivo, where the total hydroxyoctadecadienoic acid (tHODE) and 7-hydroxycholesterol (t7OHCh) are determined from the biological samples after reduction and saponification.
Polyunsaturated fatty acids and their esters are known to be quite susceptible to free-radical
mediated oxidation, while cholesterol is more resistant to oxidation. In accordance with this
chemical reactivity, it has been observed that in the case of plasma lipid peroxidation
initiated by free radical exposure, the amount of oxidation products of linoleic acid, tHODE,
generated was 30-fold higher than that of t7-OHCh. On the other hand, in cellular lipid
peroxidation experiments with different oxidative insult, cholesterol oxidation products
were detected in greater amounts than oxidation products of linoleic acid (1-3). In seleniumdeficient cells, the generation of lipid hydroperoxides was observed and cholesterol
hydroperoxide was detected as a major lipid hydroperoxide (1). The increased amounts of
t7-OHCh were also higher than those of tHODE in selenium-deficient cells (2). In contrast to
plasma oxidation, the increased amounts of cellular t7-OHCh were 5.7 times greater than
that of tHODE by free radical exposure (2). Higher levels of t7-OHCh than that of tHODE were
also observed in glutamate-treated neuronal cells (3). It should be bear in mind that the
amounts of lipid peroxidation products found in biological fluids and tissues depend upon
the rates of metabolism and clearance as well as formation. The metabolism of oxidized
cholesterol may be slower than that of the oxidized fatty acids, which may account, at least
in part, for the relatively high concentrations of oxidized cholesterol as compared to that of
the oxidized fatty acids in these experiment. Collectively, cholesterol oxidation products at 7position may be a possible biomarker of cellular lipid peroxidation.
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3rd ENOR symposium – Swansea 2013
Identification of LXR interacting proteins
Zuzana Simova, Thomas Hearn, William J. Griffiths and Yuqin Wang
Institute of Mass Spectrometry, School of Medicine, Swansea University
Liver X receptor (LXR) belongs to ligand-activated nuclear receptor superfamily and regulates
transcription of genes involved in cholesterol metabolism. Transcription is also regulated by
addition of transcription factors, coactivators and corepressors. Coactivators participate in
activation of transcription while corepressors play an important role in transcriptional
repression. The aim of our project is to look at protein complexes interacting with LXRß. This
is to be achieved by LXRß immunoprecipitation with ANTI-FLAG M2 affinity gel after LXRß is
over-expressed in HEK293T cells. Furthermore, it is well known that protein phosphorylation
or de-phosphorylation can fundamentally influence metabolism processes such as
transcription. Combining immunoprecipitation with mass spectrometry approach in a
compatible protocol should enable us to identify phosphorylation sites of enriched
coactivators and corepressors. In this approach, transcription factors and their
phosphorylation are compared in 25-Hydroxycholesterol (25-OH) treated and untreated
HEK293T cells.
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3rd ENOR symposium – Swansea 2013
Identification of Cholestenoic acids altered in motor neuron patients that
regulate motor neuron survival via Liver X Receptors
Spyridon Theofilopoulos1 *, William J. Griffiths2 *, Peter J. Crick2, Anna Meljon2,
Michael Ogundare2, Satish Srinivas Kitambi1, Andrew Lockhart3, Karin Tuschl4,
Peter T. Clayton4, Andrew A. Morris5, Adelaida Martinez6, M. Ashwin Reddy7,
Andrea Martinuzzi8, Maria T. Bassi9, Akira Honda10, Tatsuki Mizuochi11,
Akihiko Kimura11, Hiroshi Nittono12, Giuseppe De Michele13, Rosa Carbone13,
Chiara Criscuolo13, Joyce L. Yau14, Jonathan R Seckl14, Jan-Åke Gustafsson15,16,
Knut R. Steffensen16, Patrik Ernfors1, Jan Sjövall1, Ernest Arenas1 †
and Yuqin Wang2 †
*,† denotes equal contribution
1 Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and
Biophysics, Karolinska Institutet, Stockholm SE-17177, Sweden. / 2 Institute of Mass
Spectrometry, College of Medicine, Grove Building, Swansea University, Singleton Park,
Swansea SA2 8PP, UK. / 3 Translational Medicine, GlaxoSmithKline R&D China, Box No. 128,
Addenbrookes Hospital, Cambridge CB2 2GG, UK. / 4 Clinical and Molecular Genetics Unit,
UCL, Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK. / 5 Willink
Biochemical Genetics Unit, Genetic Medicine, St Mary’s Hospital, Oxford Road, Manchester
M13 9WL, UK. / 6 Barts and the London, NHS Trust, London, UK. / 7 Department of
Ophthalmology, Barts Health NHS Trust, London, UK. / 8 E. Medea Scientific Institute,
Conegliano Research Centre, Conegliano, Italy. / 9 E. Medea Scientific Institute, Laboratory of
Molecular Biology, Via D.L. Monza 20, 23842 Bosiso Parini, Lecco, Italy. / 10 Tokyo Medical
University, Ibaraki Medical Center, 3-20-1 Chuoh, Ami Ibaraki 300-0395, Japan. / 11
Department of Pediatrics and Child Health, Kurume University School of Medicine, Kurume,
Japan. / 12 Junshin Clinic, Bile Acid Institute, Tokyo, Japan. / 13 Department of Neurological
Sciences, Federico II University, Naples, Italy. / 14 Endocrinology Unit, Centre for
Cardiovascular Science, The Queen's Medical Research Institute, University of Edinburgh, 47
Little France Crescent, Edinburgh EH16 4TJ, UK. / 15 Center for Nuclear Receptors and Cell
Signaling, University of Houston, Houston, TX 77204, U.S.A. / 16 Department of Biosciences
and Nutrition, Center for Biosciences, Novum, 14183, Stockholm, Sweden.
Cholestenoic acids are intermediates in metabolism of cholesterol to bile acids and enzymes
required for their biosynthesis are expressed in the mammalian CNS. Here we describe the
cholestenoic acid profile of mammalian cerebrospinal fluid and show that specific
cholestenoic acids, including 3β,7α-dihydroxycholest-5-en-26-oic (3β,7α-diHCA) and 3βhydroxy-7-oxocholest-5-en-26-oic acids (3βH,7O-CA), activate the liver X receptors (Lxrs),
enhance Islet-1 expression and protein levels in zebrafish in vivo, and increase the number of
oculomotor neurons in the developing mouse in vitro. Mechanistically, while 3β,7α-diHCA
promoted motor neuron survival in an Lxr-dependent manner, 3βH,7O-CA promoted
maturation of precursors into Islet1+ cells. Surprisingly, a weak Lxr ligand, 3βhydroxycholest-5-en-26-oic acid (3β-HCA), caused motor neuron cell loss in vivo. Mutations
in CYP27A1 or CYP7B1, two enzymes involved in cholestenoic acid metabolism, result in two
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3rd ENOR symposium – Swansea 2013
different neurological diseases both leading to motor dysfunction, cerebrotendinous
xanthomatosis (CTX) and hereditary spastic paresis type 5 (SPG5), respectively. Analysis of
patients with SPG5 revealed an excess of the toxic Lxr ligand, 3β-HCA, while patients with
CTX and SPG5 exhibited low levels of the survival-promoting Lxr ligand, 3β,7α-diHCA in
plasma. Combined, our results show that specific cholestenoic acids selectively work on
motor neurons, via Lxr, to regulate the balance between survival and death. These findings
provide a mechanism for motor neuron dysfunction in CTX and SPG5 and suggest that efforts
aimed at restoring the balance between toxic and pro-survival Lxr ligands, such as
administration of 3β,7α-diHCA, can lead to potential treatments for motor neuron disease.
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3rd ENOR symposium – Swansea 2013
Lipids, oxidized lipids and proteins levels among Symptomatic versus
Asymptomatic
Cohen Elad, Khatib Soliman and Vaya Jacob
Oxidative Stress and Human Diseases, MIGAL-Galilee Research Institute, Kiryat Shmona and
Tel-Hai College, Israel.
Human carotid plaque components interact directly with circulating blood elements and
thus they might affect each other. We compared plaque and blood levels of oxidized lipids
including oxysterols, HDL, Paraoxonase1 (PON1) and hemoglobinA1c (HbA1c), in
symptomatic and asymptomatic patients undergoing routine endarterectomy. The lesions
were ground and extracted for lipid content and PON1 activity was determined. Oxysterols
and triglyceride levels in plaques from symptomatic and asymptomatic patients did not
differ significantly, but symptomatic patients had significantly higher (135%) linoleic acid
hydroperoxide (LA-13OOH) levels in their plaque. Symptomatic versus asymptomatic also do
not differ significantly in their cholesterol and triglyceride levels in their serum, nor in their
serum PON1 activities but symptomatic patients had significantly lower (28%) serum HDL
levels and higher (18%) HbA1c levels. Thus LA-13OOH, a major atherogenic plaque element,
showed significant negative correlations with serum PON1 activity and HDL levels, and a
positive correlation with the pro-diabetic atherogenic HbA1c and thus PON1 activity and
HbA1c level in the serum may provide information of human carotid plaque stability.
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3rd ENOR symposium – Swansea 2013
Paradoxical increase in cholesterol concentrations in serum and in circulating
monocytes by plant sterol ester supplemented margarine. A prospective,
double-blind, randomized, placebo-controlled, cross-over study.
Oliver Weingärtner, Ivan Bogeski, Carsten Kummerow, Stephan Schirmer, Tim
Vanmirlo, Markus Hoth, Michael Böhm and Dieter Lütjohann
Aim: This prospective, double-blind, randomized, placebo-controlled, cross-over study was
conducted in healthy hospital employees to evaluate the effects of a 3 g plant sterol ester
supplemented margarine on cholesterol, non-cholesterol sterols and oxidative stress in
serum and in monocytes.
Methods: 16 probands, average age 34 years, with no or mild hypercholesterolemia were
randomized to a 4 week period on plant sterol ester supplemented or placebo margarine on
top of regular eating habits. After a wash-out period of one week probands switched
groups.
Results: Margarine supplemented with 3 g plant sterol esters exhibited greater plant sterol
content compared to placebo margarine (sitosterol: 102.6 vs. 1.5 µg/mg). After a 4 week
period on 3 g plant sterol ester supplemented margarine serum levels of plant sterols
increased (sitosterol: 0.26 to 0.43 mg/dl). Unexpectedly, total serum cholesterol levels
(192.3 to 218.2 mg/dl) and serum levels of lathosterol, an indicator for cholesterol synthesis,
increased as well (0.35 mg/dl to 0.44 mg/dl). These findings could be verified in isolated
circulating monocytes with increased values for plant sterols (sitosterol: 0.080 to
0.161ng/µg), cholesterol (3.98 to 6.94ng/µg) and lathosterol (0.020 to 0.039ng/µg).
Furthermore, FACS analysis revealed an increase in circulating CD14+monocytes after plant
sterol supplemented diet, but no differences in regard to the redrox state.
Conclusions: In a population of healthy hospital employees with no or mild
hypercholesterolemia regular consumption of a 3 g plant sterol ester supplemented
margarine results in a paradoxical increase in cholesterol synthesis and total cholesterol
levels in serum and in circulating monocytes.
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3rd ENOR symposium – Swansea 2013
Systematic investigation of the dependency of viral replication on the host
sterol metabolic network.
Wei Yuan Hsieh and Peter Ghazal.
Division of Pathway Medicine, University of Edinburgh
Sterols play many important roles in physiology, including maintaining cell membrane
integrity and are required for the production of vitamin D and hormones. Recent studies
suggest that the sterol metabolic network and its secondary metabolites participate in the
interferon (IFN) antiviral response. We previously showed sterol biosynthesis was downregulated upon infection in bone marrow derived macrophages (BMDM) by type one
interferon (IFN) as an antiviral response to murine cytomegalovirus (mCMV). In addition, we
reported a cellular antiviral role for macrophage production of 25-hydroxycholesterol
(cholest-5-en-3β,25-diol, 25-HC) as a component of the sterol metabolic network linked to
the IFN response. Further investigation indicates that the mevalonate-isoprenoid branch of
the sterol pathway and its intermediates play a role in the host antiviral response. However,
the precise molecular mechanisms linking IFN and 25-HC with the sterol network and the
role of the various pathway branches remain unknown. We have taken a systematic
unbiased screening using a combination of gene silencing and the use of sterol biosynthesis
inhibitors (statins). Results will be presented indicating that inhibition of Type II
geranylgeranyl branch of the sterol pathway can significantly alter viral replication,
supporting the view that protein prenylation of particular targets is involved in the viral
infection cycle. These findings underline the importance of host mevalonate-isoprenoid
pathway and its potential connections with interferon-sterol mediated antiviral effect.
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3rd ENOR symposium – Swansea 2013
Oxysterols and the Hedgehog signaling pathway
Steven Ray Wilson*, Hanne Roberg-Larsen, Nina Solberg, Petter Angell Olsen,
Martin Strand and Stefan Krauss
*Department of Chemistry, University of Oslo
Exogenous oxysterols (hydroxycholesterols) have previously been shown to be strong
agonists of the cancer-related Hedgehog (Hh) signaling pathway, but studies linking
endogenous oxysterols and Hh have been missing. In this context, we have developed and
validated a method for quantifying oxysterols in tumor cells, combining Girard T reagent
“charge-tagging”, on-line sample enrichment/reagent removal and nano liquid
chromatography. The instrumentation featured a self-cleaning on-line filtration scheme,
allowing for minimal sample preparation and high degree of automation. Method
characteristics included high precision and low detection limits (~30 fg) in  5,000 cells. Of
the studied oxysterols, endogenous 24-hydroxycholesterol (24-OHC) and 27hydroxycholesterol (27-OHC) were particularly associated with Hh activity in a dosage and
time dependent matter when cells were treated with Hh/Smoothened (SMO) antagonists.
24-OHC and 27-OHC were also up-regulated in a beta catenin (-/-) BxPC3 pancreatic tumor
cell line, suggesting a shift towards Hh-ness in the absence of the central protein of the Wnt
signaling pathway. This was hypothesis was strengthened by observation of elevated levels
of central Hh gene target and effector GLI 1, and heightened responsiveness (i.e. markedly
reduced growth rate) to Hh/SMO antagonists. Cholesterol 24-hydroxylase and cholesterol
24-hydroxylase were up-regulated, implying that 24/27-OHC were regulated on a
transcriptional level. These initial findings demonstrate a relationship between highly
quantifiable endogenous oxysterols and Hedgehog signaling.
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3rd ENOR symposium – Swansea 2013
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3rd ENOR symposium – Swansea 2013
Author index
A
D
Abdel-Khalik Jonas
Swansea University
United of Kingdom
744116@swansea.ac.uk
Diczfalusy Ulf
Karolinska Institut of Stockholm
Sweden
ulf.diczfalusy@ki.se
Abrescia Paolo
University of Naples Federico II
Italy
paolo.abrescia@unina.it
Dieter Lütjohann
University of Bonn
Germany
E
Andrew Ruth
University of Edinburgh
United Kingdom
Ruth.Andrew@ed.ac.uk
El Kharrassi Youssef
Université Hassan I
Morocco
Arenas Ernest
Karolinska Institute of Stockholm
Sweden ernest.arenas@ki.se
B
Baron Silvère
Université Blaise Pascal
Clermont-Ferrand - France
silvere.baron@univ-bpclermont.fr
Björkhem Ingemar
Karolinska Institutet
Sweden
ingemar.bjorkhem@karolinska.se
Bydlowski Sergio
University of São Paulo
Brasil
spbydlow@usp.br
C
G
Ghazal Peter
University of Edinburgh
United of Kingdom
p.ghazal@ed.ac.uk
Griffiths William
Institute of Mass Spectrometry
Swansea University
United of Kingdom
w.j.griffiths@swansea.ac.uk
Gondcaille Catherine
University of Burgundy
France
Catherine.gondcaille@u-bourgogne.fr
Guillou Hervé
INRA Toulouse
France
herve.guillou@toulouse.inra.fr
Cardeni Vladimiro
University of Bologna
Italy
Crick Peter
Swansea University
United of Kingdom
peter.crick@gmail.com
H
Hsieh Wei Yuan
University of Edinburgh
United of Kingdom
wayne.hsieh@ed.ac.uk
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3rd ENOR symposium – Swansea 2013
Husche Constanze
University of Bonn
Germany
constanze.husche@ukb.uni-bonn.de
Maqdasy Salwan
Clermont-Ferrand University
France
salwanmed@yahoo.com
Matysik Silke
University of Regensburg
Germany
silke.matysik@klinik.uni-regensburg.de
I
Iuliano Luigi
Sapienza University of Rome
Italy
iuliano.luigi@gmail.com
luigi.iuliano@uniroma1.it
Meljon Anna
University of Swansea
United of Kingdom
562876@swansea.ac.uk
K
Miyoshi Noriyuki
Japan
miyoshin@u-shizuoka-ken.ac.jp
Karu Kersti
karu.kersti@gmail.com
Morgan Alwena
University of Swansea
United of Kingdom
a.h.morgan@swansea.ac.uk
L
Leonarduzzi Gabriella
Italy
gabriella.leonarduzzi@unito.it
N
Leoni Valerio
Neurological Institute Besta
Italy
valerioleoni@hotmail.com
Noguchi Noriko
Lizard Gérard
University of Burgundy - INSERM
France
gerard.lizard@u-bourgogne.fr
Lovgren Sandblom Anita
anita.lovgren.sandblom@ki.se
Lütjohann Dieter
University of Bonn
Germany
dieter.luetjohann@ukb.uni-bonn.de
M
Matsyk Silke
University Hospital Regensburg
Germany
silke.matysik@klinik.uni-regensburg.de
Nury Thomas
University of Burgundy
France
thomas.nury@u-bourgogne.fr
O
Olkkonen Vesa
Helsinki
vesa.olkkonen@helsinki.
P
Poirot Marc
UMR 1037 INSERM-University Toulouse III
France
marc_poirot@hotmail.com
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3rd ENOR symposium – Swansea 2013
R
T
Rigdova Katarina
University of Swansea
United of Kingdom
669687@swansea.ac.uk
Theofilopoulos Spyridon
Karolinska Institute of Stockholm
Sweden
spyridon.theofilopoulos@ki.se
Røberg-Larsen Hanne
University of Oslo
hannero@kjemi.uio.no
V
Robertson Kevin
University of Edinburgh
United of Kingdom
kevin.robertson@ed.ac.uk
Vaya Jacob
Migal- Galilee Technology Center
Israel
vered.michal@gmail.com
Rodriguez-Estrada Maria Teresa
University of Valencia-Burjassot
Spain
Vejux Anne
University of Burgundy
France
anne.vejux@u-bourgogne.fr
S
W
Sabuco Jean-François
Sanofi – France
jean-francois.sabuco@sanofi.com
Sailer Andreas
Novartis Institutes for BioMedical Research
Switzerland
andreas.sailer@novartis.com
Saito Yoshiro
Doshisha University
Japan
ysaito@mail.doshisha.ac.jp
Simova Zuzana
University of Swansea
United of Kingdom
z.simova@swansea.ac.uk
Weingärtner Oliver
Wilson Steven Ray
University of Oslo
Norway
stevenw@kjemi.uio.no
Y
Yuguang Lin
yuguang.lin@unilever.com
Yutuc Eylan
Univeristy of Swansea
United of Kingdom
705562@swansea.ac.uk
Yuqin Wang
University of Swansea
United of Kingdom
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3rd ENOR symposium – Swansea 2013
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3rd ENOR symposium – Swansea 2013