Development of new LXR modulators that regulate LXR target genes and reduce lipogenesis in human cell models

Article abstract of DOI:10.1016/j.ejmech.2014.01.003

Four new mimics of 22-S-hydroxycholesterol (22SHC) were synthesized and evaluated using molecular modeling and tested in human muscle cells (primary myotubes) and hepatocytes (HepG2 cells). The new compounds (9, 12, 15a and 15b) showed good interrelationship between docking scores, to both LXRα and LXRβ, and in vitro results. The LXR agonist T0901317 increased the expressions of genes involved in lipogenesis (SCD1, FAS) and cholesterol efflux (ABCA1), but only 22SHC counteracted the up-regulation of SCD1 and FAS by T0901317. Compound 9 and 12 decreased the expression of SCD1, while 9 also decreased the expression of FAS. Compounds 15a showed a significant antagonistic effect on ABCA1 expression, but neither 15a nor 15b were able to counteract the effect of T0901317 on all genes examined. Lipogenesis was increased after T0901317 treatment and only 22SHC significantly counteracted this effect. Treatment with 22SHC and compound 12 reduced lipogenesis compared to control. An increased glucose uptake was observed for all compounds, except for 15b. In summary, the new synthetic 22SHC mimics showed antagonistic effects similar to that of 22SHC, but the new substances were less potent. The sulfonamide 12 showed similar effects to 22SHC and the best effect on gene expression of the new mimics, however, it was not able to reduce the effect of T0901317 as observed for 22SHC.

Full text of DOI:10.1016/j.ejmech.2014.01.003

European Journal of Medicinal Chemistry 74 (2014) 258e263
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Development of new LXR modulators that regulate LXR target genes
and reduce lipogenesis in human cell models
Ove Alexander Høgmoen Åstrand a, Ingvei Gikling b, Ingebrigt Sylte c,
,
,
*
Arild Christian Rustan b, G. Hege Thoresen b d, Pål Rongved a, Eili Tranheim Kase b
a Department of Pharmaceutical Chemistry, School of Pharmacy, University of Oslo, Oslo, Norway
b Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, P.O. Box 1068, Blindern, N-0316 Oslo, Norway
c Medicinal Pharmacology and Toxicology, Department of Medical Biology, Faculty of Health Sciences, The Artic University of Norway, Tromsø, Norway
d Department of Pharmacology, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo and Oslo University Hospital, Oslo, Norway
a r t i c l e i n f o
a b s t r a c t
Article history:
Four new mimics of 22-S-hydroxycholesterol (22SHC) were synthesized and evaluated using molecular
modeling and tested in human muscle cells (primary myotubes) and hepatocytes (HepG2 cells). The new
Received 26 April 2013
Received in revised form
3 January 2014
Accepted 4 January 2014
Available online 11 January 2014
compounds (9, 12, 15a and 15b) showed good interrelationship between docking scores, to both LXR
a
and LXR , and in vitro results. The LXR agonist T0901317 increased the expressions of genes involved in
b
lipogenesis (SCD1, FAS) and cholesterol efflux (ABCA1), but only 22SHC counteracted the up-regulation of
SCD1 and FAS by T0901317. Compound 9 and 12 decreased the expression of SCD1, while 9 also
decreased the expression of FAS. Compounds 15a showed a significant antagonistic effect on ABCA1
expression, but neither 15a nor 15b were able to counteract the effect of T0901317 on all genes examined.
Lipogenesis was increased after T0901317 treatment and only 22SHC significantly counteracted this ef-
fect. Treatment with 22SHC and compound 12 reduced lipogenesis compared to control. An increased
glucose uptake was observed for all compounds, except for 15b. In summary, the new synthetic 22SHC
mimics showed antagonistic effects similar to that of 22SHC, but the new substances were less potent.
The sulfonamide 12 showed similar effects to 22SHC and the best effect on gene expression of the new
mimics, however, it was not able to reduce the effect of T0901317 as observed for 22SHC.
Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
Lipid metabolism
Type 2 diabetes
Molecular modeling
Gene regulation
LXR modulators
1. Introduction
and storage of fatty acids [5]. Previous observations have shown
that the synthetic compound 22SHC have biological activity in
Liver X receptors (LXR
a
and LXR
b
) are key players for increased
human skeletal muscle cells, where 22SHC behaved like an LXR
antagonist in these cells. 22SHC repressed genes involved in lipo-
genesis (i.e. ChREBP, FAS, ASCL1) and lipid handling that resulted in
reduced synthesis of complex lipids even when compared to un-
treated myotubes [6]. We showed that exposure to a synthetic LXR
agonist (T0901317) increased lipogenesis more in myotubes
established from type 2 diabetics than in myotubes from lean
subjects, and that these effects could be efficiently counteracted by
22SHC. Further, 22SHC reduced de novo diacylglycerol production
below basal, reduced fatty acid uptake and oxidation at the same
time as glucose uptake and oxidation were increased, whereas
cholesterol transport was unchanged [7]. A drug with similar
metabolic properties as observed for 22SHC would represent a new
kind of drug treatment and may be an important step in the search
toward new treatments against obesity and type 2 diabetes.
Given the desirable properties of 22SHC and the importance of
having the correct stereochemistry at the 22 position [8], a series of
simpler analogs were made. The new compounds were designed to
cellular lipid accumulation and insulin resistance in skeletal mus-
cle, which is a salient feature of type 2 diabetes and metabolic
syndrome. The LXRs belong to the nuclear receptor superfamily,
which are ligand-activated transcription factors. Known endoge-
nous ligands are oxysterols (cholesterol metabolites) and glucose/
glucose-6-phosphate [1,2]. LXR is important for lipid, glucose and
cholesterol metabolism [3]. Favorable effects of activating LXR are
increased glucose uptake, increased utilization of glucose and lipids
and reduced de novo synthesis of lipids, while unfavorable effects of
LXR activation are increased accumulation of lipids in cells,
decreased insulin response and reduced glucose transport in e.g.
skeletal muscle [4].
Chronic activation of LXR in human skeletal muscle cells results
in increased glucose uptake and oxidation, and increased uptake
* Corresponding author. Tel.: þ47 22856545; fax: þ47 22854402.
E-mail address: e.t.kase@farmasi.uio.no (E.T. Kase).
0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2014.01.003

O.A. Høgmoen Åstrand et al. / European Journal of Medicinal Chemistry 74 (2014) 258e263
259
Fig. 1. Synthesis scheme for the new modulators. Experimental conditions: a) PCC, DCM, r.t. 60%. b) nBu2BOTf, DIPEA, DCM, 0 ꢀC. c) 2, DCM, ꢁ78 ꢀC, d) MeOH, H2O2, r.t. 62%. e) LiBH4,
Et2O, 0 ꢀC, 67%. f) LiAlH4, THF, 0 ꢀC, 75%. g) PBr3, CBr4, 0 ꢀC, 53%. h) 5, NaH, THF, DMF, 0 ꢀC-r.t. 48%. i) PBr3, CBr4, 0 ꢀC, 48%. j) 3, NaH, THF, DMF, 0 ꢀC-r.t. 18%. k) HNR1R2, TEA, DCM, 0 ꢀC,
20e50%. l) 5, NaH, THF, DMF, 0 ꢀC-r.t. 10e50%.
mimic 22SHC and needed to consist of the following structural
features; a cholesterol-like side chain incorporating the important
hydroxyl group with correct stereochemistry, a lipophilic linker
resembling the steroid skeleton and a polar group to mimic the
alcohol in the 3-position of 22SHC.
each compound was assessed by 1H and 13C NMR. All compounds
were also analyzed using HRMS.
The 22SHC isomer has been shown to act as an LXR antago-
nist on certain genes [6,11]. The binding affinities to the LXR
receptors were evaluated in silico using the Internal Coordinate
Mechanics (ICM) program. The results are found in Table 1 and
Few compounds have been described as selective LXR antago-
nists. One potent endogenous LXR antagonist is 5
a
,6a-epox-
the best docking pose of compound 9 docked into LXRb is seen in
ycholesterol [9], and one synthetic drug candidate in this class in
industrial pipeline is GSK2033 [10]. To our knowledge, none of the
LXR modulators currently in the pipeline address the observed
ability of 22SHC to stimulate glucose uptake and decrease lipo-
genesis and it is therefore of great interest to investigate such new
compounds.
The aim of this study was to synthesize 22SHC mimics and to
test them in vitro in human cell model systems whether our new
compounds could perform equal or better than the mother sub-
stance 22SHC on lipogenesis and on selected genes important for
lipid metabolism as well as on glucose uptake.
Fig. 2. All compounds except 15a docks similar to the parent
compound 22SHC as previously described [7,11]. The sulfon-
amide 15a orients opposite to that of 22SHC (not shown).
Compound 9 performed equally to 22SHC and the known agonist
T0901317, while compound 12, 15a and 15b only showed mod-
erate affinity for the LXR receptors. Compound 12 gave the
lowest binding affinity most likely caused by its partial docking
outside the defined binding pocket for both LXR isoforms (not
shown).
Previous studies showed that 22SHC could repress genes
involved in lipogenesis (SCD1 and FAS) in both myotubes and he-
patocytes (HepG2 cells) [7,12]. We wanted to compare the effects of
the new compounds to the effect of 22SHC on gene expression of
SCD1 and FAS in myotubes. We also explored whether the new
compounds influenced the reverse cholesterol transporter ABCA1.
The results showed that the known LXR agonist T0901317 increased
2. Results and discussion
The new LXR regulators were synthesized according to Fig.1 and
the details are listed in the Supporting information. The purity of

260
O.A. Høgmoen Åstrand et al. / European Journal of Medicinal Chemistry 74 (2014) 258e263
Table 1
predicted to bind LXRa and LXRb, but with a quite low binding
affinity. The receptor binding was confirmed by the in vitro
studies.
These results therefore indicate that simulation of the binding
affinity of potential ligands to the LXRs can be used as a tool to
predict biological activity when the compounds fit the ligand
binding pocket. Molecular modeling has also been used by others
with success to evaluate new LXR modulators [13].
Structures and corresponding docking scores for LXR
a
and LXR
Docking score
LXR LXR
b.
Structure
a
b
ꢁ17.5
ꢁ29.2
Recent data showed that 22SHC reduced de novo synthesis of
lipids also in liver cells (HepG2 cells), had no effect on intestinal
cells (CaCo2) and increased de novo synthesis of lipids in adipocytes
(SGBS cells) [7]. Therefore, we wanted to explore whether any of
the compounds tested on the expression of SCD1 and FAS could
influenced lipogenesis in myotubes and HepG2 cells. Compound 12
reduced lipogenesis in myotubes in a dose-dependent manner
(Fig. 4A). Lipogenesis in myotubes was increased after T0901317
treatment and tended to increase also in combination with test
compounds, except for 22SHC that partly counteracted this effect
(Fig. 4B). The effects of test compounds on lipogenesis were more
pronounced in HegG2 cells than observed in myotubes; T0901317
increased lipogenesis in general, except when in combination with
22SHC (Fig. 4C). 22SHC and compound 12 reduced lipogenesis
compared with control, while compound 9 only tended to do so
(Fig. 4C, p ¼ 0.1).
ꢁ25.4
ꢁ10.0
ꢁ25.6
ꢁ7.7
The mother substance 22SHC has previously been shown to
increase glucose uptake in myotubes, which is thought as a desir-
able effect [7]. Compounds 9 (Fig. 5A), 12 (Fig. 5B) and 15a (Fig. 5C)
increased glucose uptake in a dose-dependent manner, but not
compound 15b (Fig. 5D). Two of the sulfonamides, 12 and 15a, gave
ꢁ3.2
ꢁ12.3
ꢁ24.3
ꢁ13.0
ꢁ15.9
ꢁ22.3
a significant increase at 1
mM while the 2-methoxynaphtalene
analog 9 required 50
mM to give a significant increase in glucose
uptake.
3. Conclusion
The new synthetic 22SHC mimics showed antagonistic effects
similar to that of 22SHC, but the new substances were less potent.
Compound 12 showed the most similar effect and the most desir-
able profile on gene expression, however, it was not able to reduce
the effect of T0901317 to such a degree as that of 22SHC. Interest-
ingly, an increased glucose uptake was observed for all compounds,
except for 15b. The initial studies with novel 22SHC mimetics
presented in this study are promising, however, we intend to
synthesize and test new cholesterol derivatives before we do in vivo
testing of selected compounds.
the expressions of SCD1, FAS and ABCA1 as expected (Fig. 3AeC). In
concordance with previous studies [7,12], 22SHC also increased
ABCA1 expression, but had no impact on the effect on T0901317
(Fig. 3C). The compounds 9 and 12 decreased the expression of
SCD1 (Fig. 3A), while only 9 decreased the expression of FAS
(Fig. 3B). Compound 15a and 15b had no effects on the genes tested
with the exception that 15a down-regulated the expression of
ABCA1 (Fig. 3C).
For a compound to induce a biological activity, the compound
must stabilize the receptor structure in a proper conformation,
and it is not necessarily a direct relationship between the receptor
binding affinity and the biological effect of the tested compounds.
Fig. 2. Compound 9 docked into the LXR
docked into the ligand-binding domain of LXR
while the amino acids near the binding pocket are shown in gray (with red and blue
representing oxygen atoms and nitrogen atoms respectively). (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of this
article.)
b
binding site. The best pose of compound 9
(PDB: 1PQ6). 9 shown in pale yellow
b
The docking predicted the receptor binding affinity for LXR
a and
LXR , while the in vitro testing measured the biological effects.
b
Compound 9 was the only compound with a docking scoring
comparable to that of 22SHC. The other compounds were also

O.A. Høgmoen Åstrand et al. / European Journal of Medicinal Chemistry 74 (2014) 258e263
261
4. Experimental
and 22(S)-hydroxycholesterol were purchased from SigmaeAldrich
(St. Louis, MO, USA). RNeasy Mini kit and RNase-free DNase were
purchased from Qiagen Sciences (Oslo, Norway). Agilent Total RNA
isolation kit was from Agilent Technologies (Santa Clara, CA, USA).
The primers were purchased from Invitrogen (Paisley, Scotland,
UK), while SYBRÒ Green and TaqManÒ reverse-transcription re-
agents kit were from Applied Biosystems (Foster City, Canada).
T0901317 was obtained from Cayman Chemical Company (Ann
Arbor, MI, USA). Hydrophobic MultiScreenÒ HTS plates were from
Millipore (Billerica, MA, USA). CorningÒ CellBINDÒ tissue culture
plates were obtained from Corning Life-Sciences (Schiphol-Rijk,
The Netherlands). The protein assay reagent was obtained from
BioRad (Copenhagen, Denmark). All other chemicals used were
standard commercial high purity quality.
4.1. Materials
Dulbecco‘s modified Eagle’s medium (DMEM-GlutamaxÔ,
5.5 mM), DMEM, foetal bovine serum, Ultroser G, penicilline
streptomycineamphotericin B, and trypsin-EDTA were obtained
from Gibco, Life Technologies (Paisley, UK). [1-14C]acetic acid
(54 mCi/mmol) and D D-glucose (6.0 Ci/mmol) were
-[14C(U)]deoxy-
purchased from ARC (American Radiolabeled Chemicals, St. Louis,
MO, USA). Insulin Actrapid was from Novo Nordisk (Bagsvaerd,
Denmark). Bovine serum albumin (BSA) (essentially fatty acid-free)
A
Treatment
+ T0901317
15
10
5
A
*
1.5
1.0
0.5
0.0
*
#
*
*
*
*
*
*
*
*
0
0
1
5
10
50
µ
Dose-response curve for 12 ( M)
B
B
Treatment
+ T0901317
6
5
4
3
2
1
0
15
*
*
*
10
5
*
*
#
*
*
0
C
C
3
2
1
0
*
60
50
40
30
*
*
*
*
*
*
*
*
20
6
#
*
*
4
2
0
*
*
*
Fig. 4. Effects of 22SHC mimics on lipogenesis in myotubes and HepG2 cells. Cells were
treated with DMSO (0.1%), 1 M T0901317, 10 M 22SHC, compounds 9, 12, 15a and
15b, for 4 days for myotubes and 24 h for HepG2 cells. The cells were incubated with
[1-14C]acetate (1
Ci/ml, 100 M) for 24 h for myotubes and 4 h for HepG2 cells before
m
m
Fig. 3. Effects of 22SHC mimics on gene expression in myotubes and HepG2 cells.
Myotubes were treated with DMSO (0.1%), 1 M T0901317, 10 M 22SHC, 10 M of
m
m
m
m
m
compounds 9, 12, 15a and 15b, for 4 days. Total RNA was isolated from the cells and
analyzed by qPCR as described in Materials and Methods. Gene expressions were
normalized to 36B4. Values represent fold change relative to control given as
means ꢂ SEM (n ¼ 3e11). Selected LXR target genes: ABCA1, ATP-binding cassette
transporter A1; FASN, fatty acid synthase; SCD1, stearoyl-CoA desaturase 1. *P < 0.05
vs. control (DMSO) and #P < 0.05 for T0901317 vs. treatment þ T0901317.
lipids were isolated by filtration through hydrophobic MultiScreenÒ HTS plates. The
levels of lipids were determined by scintillation counting. Values represent fold change
relative to control for total lipids synthesized from acetate given as means ꢂ SEM from
(A) doseeresponse for compound 12 in myotubes, n ¼ 6, (B) lipogenesis in myotubes,
n ¼ 3e6, and (C) lipogenesis in HepG2 cells, n ¼ 3 separate experiments. *P < 0.05 vs.
control (DMSO) and #P < 0.05 for T0901317 vs treatment þ T0901317.

262
O.A. Høgmoen Åstrand et al. / European Journal of Medicinal Chemistry 74 (2014) 258e263
Fig. 5. Effects of 22SHC mimics on glucose uptake in myotubes. Myotubes were treated with DMSO (0.1%), 1
Thereafter, the cells were exposed to D-[14C(U)]deoxy-D-glucose (1 M) for 4 h as described in Materials and Methods. The figures show D-[14C(U)]deoxy-D-glucose
Ci/ml, 100
uptake given as means ꢂ SEM from separate experiments. (A) 9 (n ¼ 3), (B) 12 (n ¼ 6), (C) 15a (n ¼ 3) and (D) 15b (n ¼ 3). *P < 0.05 vs. control (DMSO).
mM T0901317, 10 mM 22SHC, compounds 9, 12, 15a and 15b, for 4 days.
m
m
4.2. Docking experiment
obtained using an Autospec Ultima GCeMS (Micromass Ltd. Man-
chester, England). The MS was equipped with an electron ionization
(EI) ion source producing 70 eV electrons. The voltage scan time was
1 s and the inter scan delay time was 0.20 s. The mass spectrometer
was tuned to a resolution of 12,000. The ion source temperature was
set to 200 ꢀC and the samples were introduced into the instrument
via a direct insertion probe, which was cooled with water. The
software used was MassLynx version 4.0. (Waters, Milford, MA, USA).
The Internal Coordinate Mechanics program [14] was used for
docking of 22SHC and the newly synthesized compounds into the
ligand binding domain of the human LXR receptors. The X-ray
crystal structures of the LXR
a ligand binding domain (PDB entry:
1UHL) and LXR ligand binding domain (PDB entry: 1PQ6) were
b
used as targets for docking.
A virtual library of 22SHC and the newly synthesized compounds
was created using the ICM Chemist package. The crystal structure
was loaded, converted to an ICM object, all water molecules removed
and all hydrogen atoms optimized using the default conditions set by
the program. One of the dimers of the loaded crystal structure was
removed prior to the ligand binding pocket was identified. The vir-
tual binding pocket was created by selecting the co-crystallized
4.4. Cell culturing
4.4.1. Culturing of human myotubes
Satellite cells were isolated as previously described [15] from the
M. obliquus internus abdominis of 6 healthy donors, age 39.9
(ꢂ2.9) years, body mass index 23.5 (ꢂ1.4) kg/m2, fasting glucose 5.3
(ꢂ0.2) mM, insulin, plasma lipids and blood pressure within normal
range and no family history of diabetes. The muscle biopsies were
obtained with informed consent and approval by the National Com-
mittee for Research Ethics, Oslo, Norway. The cells were cultured in
DMEM-GlutamaxÔ (5.5 mM glucose), 2% foetal bovine serum, 2%
ꢀ
ligand and creating a ligand pocket that extends 5 A around the
ligand. The ligand was then removed and a virtual docking of the 6
compounds was performed using default conditions. The docking
scores were then calculated using the ICM Virtual Ligand Screening
add-on and the docking results are the best of three docking runs.
The processes were identical for both LXRa and LXRb.
Ultroser G, penicillin (100 units/ml) and streptomycin (100
mg/ml),
and amphotericin B (1.25 g/ml) for proliferation. At 70e80%
m
confluence the growth medium was replaced by DMEM-GlutamaxÔ
(5.5 mM glucose) supplemented with 2% foetal bovine serum, peni-
4.3. Synthesis of LXR modulators
cillin (100 units/ml) and streptomycin (100
(1.25
mg/ml), amphotericin B
The synthesis of the new LXR modulators and intermediates are
described in the Supporting information. All reagents were pur-
chased from SigmaeAldrich and used without further purification.
For column chromatography and filtration trough a pad of silica
m
g/ml), and insulin (25 pM) to induce differentiation. The cells
were cultured in humidified 5% CO2 atmosphere at 37 ꢀC, and the
medium was changed every 2e3 days. Experiments were performed
after 7 days of differentiation.
Merck silica 60 mesh (35e70 m
m) is used unless noted otherwise. 1H
and 13C NMR were recorded on Bruker DPX 300 and Bruker AVII 400
instrument equipped with a BACS-60 and a BACS-120 automatic
sample changer, respectively. All experiments were performed at
25 ꢀC in DMSO-d6 and CDCl3. High resolution mass spectrometry was
4.4.2. Culturing of HepG2 cells
The human hepatoblastoma cell line HepG2 (HB-8065, ATCC,
Manassas, VA, USA) was cultured in DMEM-GlutamaxÔ (5.5 mM

O.A. Høgmoen Åstrand et al. / European Journal of Medicinal Chemistry 74 (2014) 258e263
263
glucose) supplemented with 10% foetal bovine serum, streptomycin
(100
g/ml) and penicillin (100 units/ml) at 37 ꢀC in 5% CO2.
included in each experiment. Comparisons of different treatments
were evaluated by two-tailed, paired Student’s t-test, and P < 0.05
was considered significant.
m
4.4.3. Treatment of cells
Cells were treated with DMSO (0.1%), 1
mM T0901317, 10 mM
Acknowledgments
22SHC, compounds 9, 12, 15a and 15b, for 4 days for myotubes and
24 h for HepG2 cells, before further analyses were performed as
described in the following sections. Protein levels were measured
as previously described [16] for all wells analyzed for radiolabeled
fuel handling and no cell-detachment as sign on toxicity from any
of the treatments were detected.
This work was supported by University of Oslo. The authors
wish to thank associate professor Dag Ekeberg at the Norwegian
University for Life Sciences for grateful assistance with obtaining
HRMS data and Åse-Karine Fjeldheim for technical assistance with
the biological work.
4.5. RNA Isolation and analysis of gene expression by TaqManÒ
qPCR
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.01.003.
Myotubes were treated with DMSO (0.1%),1 mM T0901317,10 mM
22SHC, compounds 9, 12, 15a and 15b, for 4 days, harvested and
total RNA was isolated by Agilent Total RNA isolation kit (Agilent
Technologies, Santa Clara, CA, USA) according to the supplier’s total
References
RNA isolation protocol. Total RNA (1
mg/ml) was reversely tran-
[1] B.A. Janowski, P.J. Willy, T.R. Devi, J.R. Falck, D.J. Mangelsdorf, An oxysterol
signalling pathway mediated by the nuclear receptor LXRalpha, Nature 383
(1996) 728e731.
[2] N. Mitro, P.A. Mak, L. Vargas, C. Godio, E. Hampton, V. Molteni, A. Kreusch, E. Saez,
The nuclear receptor LXR is a glucose sensor, Nature 445 (2007) 219e223.
[3] E. Duplus, C. Forest, Is there a single mechanism for fatty acid regulation of
gene transcription? Biochem. Pharmacol. 64 (2002) 893e901.
[4] J.D. Davies, K.L. Carpenter, I.R. Challis, N.L. Figg, R. McNair, D. Proudfoot,
P.L. Weissberg, C.M. Shanahan, Adipocytic differentiation and liver x receptor
pathways regulate the accumulation of triacylglycerols in human vascular
smooth muscle cells, J. Biol. Chem. 280 (2005) 3911e3919.
[5] E.T. Kase, A.J. Wensaas, V. Aas, K. Hojlund, K. Levin, G.H. Thoresen, H. Beck-Nielsen,
A.C. Rustan, M. Gaster, Skeletal muscle lipid accumulation in type 2 diabetes may
involve the liver X receptor pathway, Diabetes 54 (2005) 1108e1115.
[6] E.T. Kase, B. Andersen, H.I. Nebb, A.C. Rustan, G.H. Thoresen, 22-
Hydroxycholesterols regulate lipid metabolism differently than T0901317 in
human myotubes, Biochim. Biophys. Acta 1761 (2006) 1515e1522.
[7] N.P. Hessvik, S.S. Bakke, R. Smith, A.W. Ravna, I. Sylte, A.C. Rustan,
scribed with hexamere primers using a PerkinElmer Thermal Cycler
9600 (25 ꢀC for 10 min, 37 ꢀC for 1 h, 99 ꢀC for 5 min) and a TaqMan
reverse-transcription reagents kit (Applied Biosystems). DNA
expression was determined by SYBRÒ Green (Applied Biosystems).
Primers (36B4, ABCA1, FASN, GAPDH and SCD1) were designed
using Primer ExpressÒ (Applied Biosystems). Primer sequences are
available upon request. Each target gene were quantified in dupli-
cates and carried out in a 25 ml reaction volume according to the
supplier’s protocol. All assays were run for 40 cycles (95 ꢀC for 12 s
followed by 60 ꢀC for 60 s). The transcription levels were normal-
ized to the reference control genes 36B4 and GAPDH.
4.6. Radiolabeled tracer studies
G.H. Thoresen, E.T. Kase, The liver
X receptor modulator 22(S)-hydrox-
ycholesterol exerts cell-type specific effects on lipid and glucose metabolism,
J. Steroid Biochem. Mol. Biol. 128 (2012) 154e164.
[8] E. Tranheim Kase, B. Andersen, H.I. Nebb, A.C. Rustan, G. Hege Thoresen, 22-
Hydroxycholesterols regulate lipid metabolism differently than T0901317 in
human myotubes, Biochim. Biophys. Acta Mol. Cell. Biol. Lipids 1761 (2006)
1515e1522.
4.6.1. De novo lipogenesis
Cells were treated with DMSO (0.1%), 1
22SHC, compounds 9, 12, 15a and 15b, for 4 days for myotubes and
mM T0901317, 10 mM
24 h for HepG2 cells, before exposure to DMEM supplemented with
[1-14C]acetic acid (1
mCi/ml, 100 mM) for 24 h for myotubes and 4 h
[9] T.J. Berrodin, Q. Shen, E.M. Quinet, M.R. Yudt, L.P. Freedman, S. Nagpal,
for HepG2 cells. Thereafter, cells were harvested in 0.1 M NaOH,
assayed for protein [16] and total lipids were isolated by filtration of
the cell lysate through hydrophobic MultiScreenÒ HTS plates
(Millipore, Billerica, MA, USA). The levels of lipids were determined
by scintillation counting, and lipogenesis from acetate was calcu-
lated by use of protein levels for standardization.
Identification of 5a,6a-epoxycholesterol as a novel modulator of liver X re-
ceptor activity, Mol. Pharmacol. 78 (2010) 1046e1058.
[10] W.J. Zuercher, R.G. Buckholz, N. Campobasso, J.L. Collins, C.M. Galardi,
R.T. Gampe, S.M. Hyatt, S.L. Merrihew, J.T. Moore, J.A. Oplinger, P.R. Reid,
P.K. Spearing, T.B. Stanley, E.L. Stewart, T.M. Willson, Discovery of tertiary
sulfonamides as potent liver X receptor antagonists, J. Med. Chem. 53 (2010)
3412e3416.
[11] T.A. Spencer, D. Li, J.S. Russel, J.L. Collins, R.K. Bledsoe, T.G. Consler, L.B. Moore,
C.M. Galardi, D.D. McKee, J.T. Moore, M.A. Watson, D.J. Parks, M.H. Lambert,
T.M. Willson, Pharmacophore analysis of the nuclear oxysterol receptor
LXRalpha, J. Med. Chem. 44 (2001) 886e897.
[12] E.T. Kase, G.H. Thoresen, S. Westerlund, K. Hojlund, A.C. Rustan, M. Gaster,
Liver X receptor antagonist reduces lipid formation and increases glucose
metabolism in myotubes from lean, obese and type 2 diabetic individuals,
Diabetologia 50 (2007) 2171e2180.
[13] B. Hu, E. Quinet, R. Unwalla, M. Collini, J. Jetter, R. Dooley, D. Andraka, L. Nogle,
D. Savio, A. Halpern, A. Goos-Nilsson, A. Wilhelmsson, P. Nambi, J. Wrobel, Car-
boxylic acid based quinolines as liver X receptor modulators that have LXRbeta
receptor binding selectivity, Bioorg. Med. Chem. Lett. 18 (2008) 54e59.
[14] R. Abagyan, M. Totrov, D. Kuznetsov, Icm e a new method for protein
modeling and design e applications to docking and structure prediction from
the distorted native conformation, J. Comput. Chem. 15 (1994) 488e506.
[15] M. Gaster, S.R. Kristensen, H. Beck-Nielsen, H.D. Schroder, A cellular model
system of differentiated human myotubes, Apmis 109 (2001) 735e744.
[16] M.M. Bradford, A rapid and sensitive method for the quantitation of micro-
gram quantities of protein utilizing the principle of protein-dye binding, Anal.
Biochem. 72 (1976) 248e254.
4.6.2. Glucose uptake
Myotubes were treated with DMSO (0.1%),1
22SHC, compounds 9, 12, 15a and 15b, for 4 days. Thereafter, cells
were exposed to -glucose (1 Ci/ml, 100 M) for
-[14C(U)]deoxy-
mM T0901317,10 mM
D
D
m
m
4 h. After incubation the cells were washed twice with ice-cold
phosphate buffered saline (PBS), lysed in 0.1 M NaOH, and radio-
activity measured by liquid scintillation counting. The protein
content of each sample was determined [16], and glucose uptake
was calculated using protein levels for standardization.
4.7. Presentation of data and statistical analysis
Data in text and figures are given as mean (ꢂSEM) from
n ¼ number of separate experiments. At least 3 parallels were