Cross-Talk between Fatty Acid and Cholesterol Metabolism Mediated by Liver X Receptor-
Kari Anne Risan Tobin1,
Hilde Hermansen Steineger1,
Siegfried Alberti,
Øystein Spydevold,
Johan Auwerx,
Jan-Åke Gustafsson and
Hilde Irene Nebb
Institute for Nutrition Research (K.A.R.T., H.I.N.) Institute
of Medical Biochemistry (K.A.R.T., H.H.S., O.S., H.I.N.) Institute
of Basic Medical Sciences University of Oslo N-0316 Oslo,
Norway
Center for Biotechnology (S.A., J.-A.G.)
Department of Medical Nutrition Novum, S-141 86 Huddinge,
Sweden
Institut de Genetique et Biologie Moleculaire et
Cellulaire (J.A.) 67404 Illkirch, France
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ABSTRACT
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LXR
(liver X receptor, also called RLD-1) is a
nuclear receptor, highly expressed in tissues that play a role in lipid
homeostasis. In this report we show that fatty acids are positive
regulators of LXR
gene expression and we investigate the molecular
mechanisms underlying this regulation. In cultured rat hepatoma and
primary hepatocyte cells, fatty acids and the sulfur-substituted fatty
acid analog, tetradecylthioacetic acid , robustly induce LXR
(up to 3.5- and 7-fold, respectively) but not LXRß (also called OR-1)
mRNA steady state levels, with unsaturated fatty acids being more
effective than saturated fatty acids. RNA stability and nuclear run-on
studies demonstrate that changes in the transcription rate of the
LXR
gene account for the major part of the induction of LXR
mRNA
levels. A similar induction of protein level was also seen after
treatment of primary hepatocytes with the same fatty acids. Consistent
with such a transcriptional effect, transient transfection studies with
a luciferase reporter gene, driven by 1.5 kb of the 5'-flanking region
of the mouse (m)LXR
gene, show a peroxisome
proliferator-activated receptor-
-dependent increase in luciferase
activity upon treatment with tetradecylthioacetic acid and the
synthetic peroxisome proliferator-activated receptor-
activator, Wy
14.643, suggesting that the mLXR
5'-flanking region contains the
necessary sequence elements for fatty acid responsiveness. In addition,
in vivo LXR
expression was induced by fatty acids,
consistent with the in vitro cell culture data. These
observations demonstrate that LXR
expression is controlled by fatty
acid signaling pathways and suggest an important cross-talk between
fatty acid and cholesterol regulation of lipid metabolism.
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INTRODUCTION
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Both primitive and complex organisms have integrated systems by
which gene expression is adapted in response to changes in intake of
basic dietary components such as carbohydrates or lipids. Two classical
examples of such transcriptional control systems governing gene
expression by lipids in higher organisms are the peroxisome
proliferator- activated receptors (PPARs), members of the nuclear
hormone receptor superfamily, and the sterol-regulatory element-binding
proteins (SREBPs), a subgroup of helix-loop-helix transcription
factors. PPARs have a pivotal role in regulation of intermediary
metabolism, both in liver and adipose tissue, where they control the
expression of several genes associated with fatty acid metabolism
(reviewed in Refs. 1, 2, 3, 4, 5). Conversely, fatty acids or their metabolites
are not only reported to be natural ligands for the different PPARs
(reviewed in Refs. 2, 3, 4, 5), but they can also control the expression of
these receptors, as demonstrated for PPAR
(6). SREBPs, synthesized
as membrane-bound precursors, are activated by proteolytic cleavage
upon sterol depletion to generate a transcriptionally active
NH2-terminal fragment (reviewed in Ref. 7).
Interestingly, SREBPs not only control cholesterol metabolism (reviewed
in Ref. 7), but are also involved in the control of fatty acid and
triglyceride metabolism (8, 9, 10, 11, 12, 13, 14).
Although clearly important for the transcriptional control of gene
expression by sterols and fatty acids, SREBPs and PPARs are not the
only transcription factors which are modulated by intermediary
metabolic compounds. Another prototypical example of such a
transcription factor is the liver X receptor
[LXR
, also called
RLD-1 (15, 16)] the activity of which is stimulated by several
metabolic products in cholesterol, steroid hormone, and/or bile acid
metabolic pathways (16, 17, 18, 19), including compounds like mevalonate, and
naturally occurring oxysterols, such as 22(R)-hydroxycholesterol,
24(S)-hydroxycholesterol, and 24,25(S)-epoxycholesterol (17, 18, 19).
Interestingly, an LXR response element (LXRE) was identified in the
promoter of the rat 7
-hydroxylase gene, the rate-limiting enzyme in
the conversion of cholesterol into bile acids (16, 19). Further
evidence supporting an important role of LXR
in lipid homeostasis
was provided by the loss of capacity to regulate catabolism of dietary
cholesterol in mice in which the LXR
gene was made nonfunctional by
homologous recombination, an effect for which the isoform, LXRß,
could not compensate (20). This suggests that the two isoforms, LXR
and LXRß, do not have overlapping functions. They also have
differential expression patterns: LXRß is ubiquitously expressed,
whereas LXR
is restricted to metabolically active tissues, such as
liver, kidney, intestines, and the adrenal glands.
In view of the important cross-regulation between sterol and fatty acid
metabolism described for SREBP and PPAR
, we were interested in
determining whether a similar regulation by metabolites from lipid
metabolism occurs for LXR
. We therefore analyzed whether LXR
expression is modified by fatty acids in the liver. Our results suggest
that LXR
gene expression is modulated by fatty acids in a complex
fashion, involving both increased transcription rates and mRNA
stability. These data suggest an important cross-talk between fatty
acid and cholesterol metabolism mediated by LXR
.
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RESULTS
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The Effects of Fatty Acids on LXR
and LXRß mRNA Levels in Rat
Hepatoma Cells in Culture
We have previously shown that fatty acids are able to induce the
levels of PPAR
and RXR
mRNA in hepatoma cells and cultured
hepatocytes (6, 21, 22). LXR
has been shown to have the same tissue
distribution as PPAR
and RXR
(1, 23). In addition, LXR
is
thought to be an important regulator of lipid metabolism
[i.e. in cholesterol, steroid hormone, and bile acid
catabolic pathways (20)]. Here we investigate the possibility that
fatty acids regulate the expression of LXR
and LXRß, and we
therefore measured the levels of their respective mRNAs by Northern
blot analysis (Table 1
and Fig. 1
, A and B). 7800C1 rat hepatoma cells
were treated for 3 days with different fatty acids (C14:0, C18:0,
C18:1, C18:3) and the sulfur-substituted fatty acid analog,
tetradecylthioacetic acid (TTA) (Table 1
). With nonmodified fatty
acids, only slight inductions of the LXR
mRNA level were observed
(for instance, 2-fold induction with C18:3). However, TTA resulted in a
3.5-fold induction of LXR
mRNA. The mRNA level of LXRß was
unchanged by all the treatments indicated (Table 1
).
Since TTA was the most effective inducer of peroxisomal
ß-oxidation in 7800C1 hepatoma cells (24, 25, 26, 27) and was the strongest
inducer of LXR
mRNA level in rat hepatoma cells, this fatty acid
analog was further used to investigate the kinetics of the LXR
mRNA
level. The 7800C1 hepatoma cell line has previously been shown to
maintain liver-specific functions in vitro under controlled
experimental conditions (24, 25). In addition, the hepatoma cells do
not, in contrast to hepatocytes, undergo time-dependent phenotypic
changes. We have shown earlier that these cells express PPAR
,
glucocorticoid, and insulin receptors and possess metabolic activities
such as peroxisomal ß-oxidation similar to rat hepatocytes (6, 27, 28). The cells were hence treated with 50 µM
TTA for up to 72 h, and total RNA was prepared and subjected to
Northern blot analysis (Fig. 1
, A and B). The major inductions due to
treatment of TTA occurred during the first 8 h of treatment with
further increase up to 24 h (Fig. 1
, A and B). A decline in the
LXR
mRNA level was observed after 72 h (Fig. 1
, A and 1B).
LXRß expression was not significantly affected by these treatments
(Fig. 1
, A and B).
Fatty Acids Increase LXR
mRNA Stability and Transcription
Rate
To further define the mechanism underlying the elevated LXR
steady state mRNA level observed after fatty acid administration, we
tested whether TTA treatment affected the stability of LXR
or LXRß
mRNAs. 7800C1 hepatoma cells were treated with TTA (50
µM) for 72 h. The transcriptional inhibitor
actinomycin D (2.5 µg/ml) was then added and cells harvested at
different time points within a period of 10 h. mRNA transcription,
relative to control, was plotted against time, and the half-lives of
the transcripts were estimated by extrapolation in the linear part of
the mRNA time curve (Fig. 2A
).
Actinomycin D inhibited the LXR
mRNA synthesis and prevented further
induction by TTA. TTA led to an increase in the half-lives of LXR
transcripts as compared with the control (5.8 and 4.0 h,
respectively; Fig. 2B
). LXRß mRNA stability remained constant under
all conditions tested (Fig. 2
, A and B). These results indicate that
the increase in the steady state levels of mRNA for LXR
in 7800C1
hepatoma cells after TTA treatment is at least partially due to a
stabilization of the LXR
mRNA.
The stabilization of LXR
mRNA is, however, insufficient to
explain the marked increase in the LXR
mRNA steady state level after
treatment with TTA. We therefore performed nuclear run-on studies to
determine the transcription rate after treatment with TTA. Figure 2C
shows the results from a representative run-on experiment. This
experiment was repeated and similar results were obtained. In nuclei
from 7800C1 hepatoma cells treated for 2, 4, and 6 h with TTA (50
µM), the transcription rates of LXR
increased after 2
and 6 h of stimulation by a factor of 2.5 and 11.6, respectively.
Only a slight up-regulation of the transcription rate of LXRß
mRNA was obtained after 2 and 6 h (2.5 and 3.5, respectively),
whereas the transcription of the human ribosomal protein L27 (control)
gene did not change (Fig. 2C
). The empty vector backbone for the LXR
and LXRß cDNAs were used as negative controls. These results indicate
that the up-regulated level of LXR
mRNA expression is mainly due to
an increased transcriptional rate, with some additional contribution
from the slightly enhanced mRNA stability.
Fatty Acid Regulation of LXR
mRNA Levels in Primary Rat
Hepatocyte Cultures
To study how fatty acids induce the LXR
mRNA level in a more
physiological system, we treated primary rat hepatocytes in culture
with different fatty acids. The rat hepatocytes were treated for
24 h with C18:1, C18:3, C20:4, C20:5, C22:6, and TTA (Fig. 3
, A and B). The hepatocytes treated with
different fatty acids and TTA showed a robust increase (4- to 7-fold
depending on fatty acid) in steady state LXR
mRNA levels after
24 h relative to untreated cells (Fig. 3
, A and B). Again, the
levels of LXRß mRNA were not affected by any of the fatty acids
tested (Fig. 3
, A and B).

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Figure 3. Effects of Unsaturated Fatty Acids on LXR
and LXRß mRNA Levels in Cultured Rat Hepatocytes after Treatment for
24 h
A, Autoradiograms showing the mRNA level of LXR and
LXRß in cultured primary rat hepatocytes treated with 1
mM oleic acid (C18:1), 1 mM linolenic acid
(C18:3), and 50 µM TTA for 24 h as described in
Materials and Methods. The figure shows representative
results from experiments repeated three times. B, The mRNA values for
LXR ( ) and LXRß ( ) were measured after stimulation of
cultured hepatocytes by different fatty acids. The concentrations of
the fatty acids were: 1 mM oleic acid (C18:1), 1
mM linolenic acid (C18:3), 0.3 mM arachidonic
acid (C20:4), 0.3 mM EPA (C20:5), 0.3 mM DHA
(C22:6), and 50 µM TTA. The figure shows the calculated
relative mRNA values (control = 1) after scanning of the
autoradiograms (see Materials and Methods), and results
are expressed as the mean ± SEM from three
independent experiments.
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Fatty acid treatment did not cause alterations in the general gene
expression pattern in rat hepatocytes, as evidenced by the absence of
significant changes in the mRNA levels of the retinoic acid receptor
(RAR
) (data not shown) and the ribosomal protein L27 (Fig. 3A
),
as well as shown in earlier studies for RXRß (21). Taken together,
our results show that LXR
gene expression is strongly regulated by
fatty acids, while interestingly the isoform LXRß mRNA level appears
to be much less dependent on these fatty acids.
Fatty Acid Regulation of LXR
Protein Levels in Primary Rat
Hepatocyte Cultures
After studies of the fatty acid-induced mRNA expression of LXR
in primary rat hepatocytes, we studied whether the induction could also
be seen at protein level. LXR
protein was monitored in liver protein
extracts by immunoblotting. Antisera against LXR
specifically
recognized a band at 51 kDa, which is in agreement with its calculated
mol wt (Fig. 4A
)(15). LXR
protein
level was induced up to 3.2-fold in cultured hepatocytes treated with 1
mM linolenic acid (18:3) for up to 48 h, relative to
control cells (Fig. 4
B). The monounsaturated fatty acid oleic acid
(18:1) induced LXR
protein level 2.3-fold and linolenic acid
1.8-fold in cultured hepatocytes 24 h after stimulation (Fig. 4C
).
These results are in concordance with the fatty acid regulation of
LXR
mRNA shown in Fig. 3
, A and B. Treatment of primary hepatocytes
with TTA and Wy 14.643, a synthetic PPAR
activator, gave only minor
induction of LXR
protein.
The 5'-Flanking Region of the Mouse (m)LXR
Gene Confers
Responsiveness to Fatty Acids
To examine the upstream region of the mLXR
gene for sequences
that might mediate the transcriptional effect of fatty acids on LXR
gene expression, a luciferase reporter gene construct containing part
of the 5'-flanking region of LXR
gene was used. About 1,500 bp of
the LXR
5'-flanking sequence (LXR
(-1500/+1800)LUC) were
subcloned in the correct orientation upstream of the firefly
luciferase-encoding sequence in the plasmid pGL3-basic (Materials
and Methods). This reporter construct was transiently transfected
into COS-1 cells in the presence of an expression vector encoding
RXR
(pCMV-RXR
). Stimulation with increasing doses of either TTA
or the specific PPAR
activator, Wy 14.643, gave only minor effects
on luciferase activity (Fig. 5A
).
Cotransfection of PPAR
expression plasmid (pSG5-PPAR
) in addition
to RXR
expression vector without any stimulation gave 2.6-fold
induction, indicating the presence of endogenous ligands for PPAR
in
COS-1 cells. However, cotransfection of PPAR
together with
increasing doses of either TTA or Wy 14.643 induced the reporter gene
activity up to 5.5-fold for TTA, and 4-fold for Wy 14.643 compared with
unstimulated cells (Fig. 5A
). Concentrations above 100
µM TTA and 150 µM Wy
14.643 was toxic to the cells. These observations indicate that PPAR
might be a possible candidate for mediating the fatty acid effect. In a
similar manner, both linolenic acid and bezafibrate stimulated
luciferase activity (data not shown).
Next, different 5'-deletion constructs of the LXR
5'-flanking
region were transfected into COS-1 cells. The PPAR
-dependent TTA
induction was still present for constructs deleted to -700 and -100
bp, but the reporter gene activity was reduced from 9.5-fold to 6-fold
[LXR
(-700/+1800)LUC] and 5-fold
[LXR
(-100/+1800)LUC], and finally down to 2.5-fold for a
construct [LXR
(-100/+125)LUC] where much of the 3'-part is
deleted. This suggests the presence of one or more PPAR-responsive
elements (PPREs) in the 5'-flanking region of the mLXR
gene (Fig. 5B
).
These results indicate the existence of cis-acting sequences
mediating the fatty acid action, probably constituting PPREs.
Computer-based analysis of the 5'-flanking region of the mLXR
gene
indicates the presence of several sequence elements with a good
homology to the consensus PPRE (Fig. 5C
).
These elements need to be further examined in future studies.
Effect of Polyunsaturated Fatty Acids (PUFAs) and PPAR
Agonists
on the LXR
mRNA and Protein Levels in Vivo
To establish the relevance of these in vitro
observations, we examined the effect of PUFAs on liver LXR
mRNA and
protein levels in vivo, where rats were fed a high-fat diet
(15% soy oil) for 48 h. Northern analysis showed that the PUFA
diet induced the liver LXR
mRNA level 3-fold (Fig. 6A
, upper panel), whereas
semiquantitative immunoblot analysis of LXR
protein from rat liver
showed a 3.3-fold increase after feeding with the PUFA diet (Fig. 6A
, lower panel).
Rats were then given TTA, Wy 14.643, or linolenic acid by gastric
intubation for 3 days (Materials and Methods). Liver LXR
mRNA in animals given different PPAR
activators was induced
approximately 2-fold for TTA and Wy 14.643, but to a lower degree by
linolenic acid (Fig. 6B
, upper panel), whereas LXR
protein expression was induced 3-fold by the potent PPAR
-agonist Wy
14.643, but also by TTA and linolenic acid (1.8-fold and 2.2-fold,
respectively)(Fig. 6B
, lower panel).
Taken together, feeding rats a diet rich in unsaturated fatty
acids, or other PPAR
activators, results in an induction in LXR
mRNA and protein level. These data are concordant with the observed
changes in LXR
mRNA and protein levels after fatty acid treatment of
rat hepatocyte and hepatoma cultures (Figs. 1
, 3
, and 4
).
Effects of Fasting-Refeeding on LXR
mRNA Steady State Levels
Finally, we investigated whether the LXR
gene is regulated by
nutritional intake in a physiological setting by using the
fasting-refeeding model. Fasting of rats for 24 h increased the
LXR
mRNA steady state level by approximately 3-fold (Fig. 7
). After refeeding the mRNA level
returned to normal levels after 1 day. As a verification that the
animals were in a proper fasted/refed state, we analyzed plasma glucose
and plasma insulin levels. Fasting resulted in an expected decrease of
plasma glucose (from
9 nmol/liter to 6 nmol/liter) and insulin (from
2.3 ng/ml to 0.5 ng/ml), and both glucose and insulin values
returned to normal after 1 day of refeeding (data not shown).

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Figure 7. Effects of Fasting-Refeeding on LXR mRNA Steady
State Levels
Rats were fasted for 24 h and were allowed free access to food for
24 h following the fast. Total hepatic RNA was prepared and used
to detect LXR mRNA by Northern blotting (see Materials and
Methods). The figure represents data from one experiment and
has been repeated with similar results.
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DISCUSSION
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Transcriptional control of gene expression is a common
mechanism by which lipids as well as other nutrients affect metabolism.
The key transcription factors known to be involved in mediating control
of gene expression by lipids belong to the PPAR and SREBP families of
transcription factors (reviewed in Refs. 1, 2, 3, 4, 5, 6, 7). Whereas the activity of
SREBP is modulated by cholesterol and its metabolites, the various
PPARs seem to be activated upon binding of fatty acids and fatty
acid-derived metabolites, such as prostaglandins or leukotrienes
(reviewed in Refs. 1, 2, 3, 4, 5, 6, 7). The nuclear receptor LXR
was recently
shown to be a transcriptional modulator capable of responding to
changes in levels of cholesterol metabolites, suggesting that it could
be a pivotal cholesterol sensor (16, 18, 19, 20). In this manuscript we
demonstrate that in the liver, LXR
expression is induced by fatty
acids. This effect seems to be the consequence of a direct induction of
LXR
gene transcription, mediated through putative fatty acid
response elements located in the proximal LXR
5'-flanking region.
Since this regulation was observed both in cultured primary hepatocytes
in vitro as well as in vivo in the intact animal,
we believe this regulation has direct physiological relevance in the
control of lipid metabolism. This is further supported by the
observation that rats fasted during a period of 24 h show an
increase in LXR
mRNA level. It has been shown earlier that there is
an increase of plasma FFA during fasting (29), which could possibly
mediate the up-regulation of LXR
gene expression observed. Recently
it was suggested that PPAR
has a role in the transcriptional
response to fasting, since fasting induces several PPAR
target genes
encoding enzymes involved in the fatty acid oxidative pathway, an
effect abolished in PPAR
-/- mice (30, 31).
The transcription factors involved in mediating this effect of fatty
acids on LXR
expression are currently unknown. In view of the well
established capacity of fatty acids to serve as ligands for the PPARs,
these nuclear receptors are prime candidates. Consistent with this
notion is the fact that several potential PPRE-like sequences are
located throughout the LXR
5'-flanking region. Our laboratories are
at present trying to identify the cis-acting sequence
elements involved in mediating this response.
Classically it was believed that fatty acid and cholesterol
biosynthesis and catabolism occurred along distinct biochemical
pathways with relatively little interaction. In general, when the
organism senses low cholesterol level, SREBP is activated and
up-regulates a number of genes involved in cholesterol synthesis such
as hydroxymethylglutaryl-coenzyme A reductase (7). From various
physiological conditions as well as from a number of disease states it
appears, however, that fatty acid and cholesterol metabolism are
coregulated and intricately intertwined (7). A good example of such
cross-talk is the controlling action that the SREBP transcription
factor family exerts on both cholesterol and fatty acid metabolism.
SREBP directly controls the expression of a set of genes involved in
fatty acid and triglyceride metabolism, such as the genes for
lipoprotein lipase (LPL) (9, 13), acetyl coenzyme A carboxylase (12, 13), and fatty acid synthetase (FAS) (8, 9, 13). Through this action
SREBP indirectly controls the generation of natural activators and
ligands for another lipid-controlled transcription factor, PPAR (11).
In addition to controlling the production of fatty acid-derived PPAR
ligands, SREBP was recently shown to directly induce transcription of
the PPAR
gene (11, 32). Certain fatty acids, the end products of the
enzymatic pathways SREBP and PPAR
are involved in, potentiate the
SREBP-regulated gene transcription (33, 34).
In addition, LXR
itself has been demonstrated to affect the
expression of various genes involved in fatty acid metabolism, such as
stearoyl-CoA desaturase, fatty acid synthase, acetyl CoA carboxylase,
and SREBP-1 (20). The regulation of the expression of the cholesterol
sensor LXR
by fatty acids indicates another important point of
cross-talk between these two chemically distinct classes of lipids,
i.e. fatty acids and cholesterol.
This cross-regulation leads us to hypothesize that when the
organism is challenged with an increased lipid load, usually composed
of both fatty acids and cholesterol, an integrated response is mounted
allowing it to handle this challenge. Triglycerides and fatty acids
derived from them are evolutionarily considered as excellent energy
sources, and therefore fatty acids are used either as direct substrates
for ß-oxidation or stored as an energy reserve in the adipocytes
(reviewed in Refs. 1, 2, 3, 4, 5, 6, 7). Both of these potential pathways are
stimulated by a feed-forward regulatory loop, controlled by the PPAR
family of fatty acid- activated nuclear receptors. In fact, activation
of PPAR
by fatty acids, primarily in liver and muscle, enhances
energy production through its stimulating effects on the ß-oxidation
pathways, whereas PPAR
activation by fatty acids increases storage
of excess fatty acids in the form of triglycerides in adipocytes. High
cholesterol levels, in contrast to fatty acids, are potentially toxic,
and therefore an intricate control circuitry exists to keep cholesterol
levels in balance (20). As underscored by our data, fatty acids will
not only enhance PPAR activity (feed-forward loop), but they will also
induce LXR
gene expression (cross-regulatory loop). In addition,
cholesterol in food provides potential ligands and activators of LXR
receptor. Through induction of LXR
levels (via fatty acids) and
through the enhanced supply of its cholesterol-derived ligands, the
LXR
-regulatory pathway facilitates the elimination of excess
cholesterol by feed-forward stimulation of its conversion to bile
acids. In addition, cholesterol buildup, through inhibition of the
proteolytic cleavage of SREBP, also prevents any further de
novo synthesis or uptake of additional cholesterol. The final
result of this integrated control circuitry, involving both fatty acids
and cholesterol, is an optimal energy utilization and a tight control
of cholesterol levels both at the intra- and extracellular level.
In conclusion, our data document transcriptional control of LXR
expression by fatty acids and suggest that this allows cross-talk
between gene regulation by fatty acids and cholesterol,
respectively.
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MATERIALS AND METHODS
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Materials
Hams F-10 medium and horse and calf serums were from Flow
Laboratories (Irvine, UK). Anti-pleuropneumonia-like organisms,
fungizone, penicillin, and streptomycin, were from Life Technologies, Inc.(Gaithersburg, MD). TTA (C14-S-C2) was
synthesized as previously described (24). Guanidium isothiocyanate was
obtained from Merck & Co., Inc.(Hohenbrunn, München,
Germany). Agarose was purchased from Bio-Rad Laboratories, Inc. (Richmond, CA). Restriction endonucleases and protease
inhibitor (Complete Protease Inhibitor Cocktail Tablets) were purchased
from Roche Molecular Biochemicals (Mannheim, Germany).
Multiprime DNA labeling systems, radiolabeled
[
-32P]dCTP, Hybond-C-Extra nitrocellulose
membrane, and the ECL Western Blotting Kit were purchased from
Amersham Pharmacia Biotech (Buckinghamshire, UK).
Bio-Trans nylon filter was from ICN Biochemicals, Inc.
(Irvine, CA). Polyclonal antibody against LXR
was purchased from
Santa Cruz Biotechnology, Inc. (no. SC-1206, Santa Cruz,
CA. A cDNA probe of the human ribosomal protein L27 ( ATCC
no. 107385) was purchased from ATCC (Manassas, VA).
pGL3-basic luciferase reporter vector was obtained from Promega Corp. (Madison, WI). Other chemicals, including Wy 14.643 and
fatty acids, were obtained from Sigma (St. Louis, MO).
Animals
All animal use was approved by the Norwegian Animal Research
Authority (NARA) and registered by the authority. Male Wistar rats of
approximately 250 g were maintained in cages at 23 C in rooms with
lights on from 0800 h2000 h. All rats had free access to water and a
standard commercial low-fat diet (2.9% wt/wt) if not otherwise stated.
In one experiment, rats were fed a standard diet containing 15% soy
oil (PUFAs) (35). The control group was allowed free access to standard
commercial low-fat diet (2.9% wt/wt). In the fasting studies, the rats
were deprived of food for 24 h after which one group was killed
for liver excision, and another group was allowed free access to
standard laboratory chow for 24 h before death. In another
experiment, rats were given different PPAR
activators by gastric
intubation once a day for 3 days. The PPAR
activators given were TTA
(50 mg once a day), Wy 14.643 (1 mg once a day), and linolenic acid (90
mg once a day) dissolved in 2% carboxymethyl cellulose (CMC). At the
end of the experiment, animals were killed and livers rapidly frozen in
liquid nitrogen and stored at -70 C until RNA or protein could be
isolated.
Cell Culture, Transient Transfections, and Luciferase
Assays
The establishment, cloning, and propagation of Morris hepatoma
7800C1 cells have been described previously (36). Cells were cultured
as monolayers in 140 x 20-mm culture dishes (Greiner,
Kremünster, Austria) and plated at 24 x
105 cells per dish in F-10 medium with 10% horse
serum and 3% FCS. Hepatocytes from male rats were isolated by the
method of Berry and Friend (37) with modifications described by Seglen
(38). Culture conditions were as reported previously, and cell
treatment during the experimental period was the same as for hepatoma
cells. Monkey kidney COS-1 cells (ATCC no. CRL 1650) were
grown in DMEM supplemented with 10% FBS. Growth medium for all cells
was supplemented with penicillin (50 U/ml), streptomycin (50 µg/ml),
fungizone (2.5 µg/ml), and anti-pleuropneumonia-like organisms (50
µg/ml). The incubation conditions were 37 C in a humidified
atmosphere of 5% CO2 and 95%
O2. Medium and additions were renewed every
48 h and always 24 h before harvesting the cells. Transient
transfections of COS-1 cells were performed in 30-mm tissue dishes at a
density of 2 x 105 cells per well after the
calcium phosphate precipitation method essentially as described in Ref.
39 . TTA was dissolved in alcalic water, and Wy 14.643 was
dissolved in Me2SO before addition to the
transfection medium at appropriate concentrations. Each well received 5
µg test plasmid and 5 µg ß-galactosidase plasmid as internal
control, and 0.4 µg of pCMV-RXR
or pSG5-PPAR
expression vectors
in the experiments stated. Cells were harvested, cytosol extracts were
prepared, and luciferase activities were measured according to the
Promega Corp. protocol. Results were normalized against
ß-galactosidase activity measured by incubating 10 µl extract with
0.28 mg
o-nitrophenyl-ß-D-galactopyranoside
(ONPG) in 50 mM phosphate buffer, pH 7.0, 10
mM KCl, 1 mM
MgCl2 for 30 min at 30 C and reading absorbance
at 420 nm.
Cell Treatment
Fatty acids; TTA (50 µM); myristic acid (C14:0) (1
mM); stearic acid (C16:0) (1 mM); oleic acid
(C18:1) (1 mM); linolenic acid (C18:3) (1 mM);
arachidonic acid (C20:4) (0.3 mM); eicosapentaenoic acid
(EPA) (C20:5) (0.3 mM) and docosahexaenoic acid (DHA)
(C22:6) (0.3 mM) were added to cell cultures during the
experimental period from 4 to 72 h. Fatty acids were added as a 4
mM stock solution dissolved in 6% fatty acid-free BSA to
the cells. The concentration of TTA (50 µM) was chosen on
the basis of maximal induction of peroxisomal acyl-CoA oxidase enzyme
activity both in hepatoma cells and hepatocytes in culture (24). The
concentration of fatty acids was based on dose-response experiments
(data not shown)(6, 21). The concentration of arachidonic acid, EPA,
and DHA (0.3 mM) was based on the fact that the addition of
1 mM concentration of these long unsaturated fatty acids
was toxic to the rat hepatocytes based on the Trypan-Blue-Exclusion
test. In addition, dose-response experiments performed by Tollet
et al. (40) in cultured hepatocytes , showed that 0.3
mM arachidonic acid and EPA resulted in maximal
induction of cytochrome P4504A1 mRNA levels.
cDNA and Reporter Constructs
The cDNA for human ribosomal protein L27
(ATCC no. 107385), rat LXR
(15), rat LXRß (41), and
glyceraldehydes-3-phosphate dehydrogenase (42) were used as probes in
Northern hybridizations. Murine LXR
5'-flanking reporter construct
was made by subcloning the 5'-upstream regulatory sequence between
-1500 bp and +1800 of the mLXR
gene into the pGL3-basic luciferase
reporter plasmid to yield the pLXR
(-1500)LUC reporter construct.
This construct contains the 2 first exons with the intervening intron,
in addition to the 5'-flanking region of the gene (1535 bp upstream of
exon 1). A complex multiple transcription start site is located in exon
1, and the translation start site is located in exon 2. The segment is
fused to luciferase 320 bp downstream of exon 2. A detailed description
of the cloning and characterization of the mLXR
gene is described
elsewhere (43). Deletion constructs were made by exonuclease III
digestion. The constructs was verified by restriction enzyme analysis
followed by partial DNA sequencing (ABI Prism Dye Terminator Cycle
Sequencing, Perkin Elmer Corp., Norwalk, CT).
Preparation and Analysis of RNA
Total RNA from 7800C1 hepatoma cells and hepatocytes was
extracted by the guanidium thiocyanate method (44), whereas total RNA
from liver tissue was extracted by using Trizol Reagent for total RNA
extraction (Life Technologies, Inc.). Northern blot
analysis of RNA was performed as described earlier (25).
[
-32P]dCTP-labeled cDNA probes were prepared
using a standard multiprime DNA-labeling kit (RPN 1601Y, Amersham Pharmacia Biotech, Buckinghamshire, UK). Specific activities of
26 x 108 cpm/µg DNA were obtained.
Semiquantitative results of the blots were obtained by scanning of
autoradiograms using an XRS 3 sc scanner and the Bio Image System from
Millipore Corp. (Bedford, MA) showing linear increments
within the working range used (530 µg RNA). For statistical
analysis of the mRNA results, the mean control values were set equal to
unity, and variation within the group was calculated accordingly.
Corresponding relative values (mean ± SEM) were
calculated for the experimental groups.
Nuclear Run-On Transcription and mRNA Stability Assays
The nuclear run-on assay was performed as described by Andersson
et al. (45). The probes used were cDNAs of rat LXR
[cloned into pGEMT (15)], rat LXRß (cloned into pBluescript
(SK+)(41)] and the human ribosomal protein L27
[cloned into pBluescript (SK+)]. The empty
vectors into which the cDNAs were cloned [pBluescript
(SK+) and pGEM-T vector] were used as
controls.
For mRNA stability studies, 7800C1 Morris hepatoma cells were treated
with TTA (50 µM) for 3 days. The incubation was continued
in the presence of actinomycin D for maximally 12 h, and cells
were harvested at different time points during this period. The
relative mRNA transcription relative to control was plotted in a
time-curve, and the half-lives of the transcripts were estimated by
extrapolation in the linear part of the mRNA time curve. The
concentration of actinomycin D used in this experiment (2.5 µg/ml)
inhibited incorporation of [3H]-uridine into
RNA by more than 95% after 0.5 h (data not shown).
Immunoblotting
Liver tissue was homogenized in PBS containing 1% NP-40, 0.5%
sodium deoxycholate, 0.1% SDS, and protease inhibitors (Complete
Protease Inhibitor Cocktail Tablets, Roche Molecular Biochemicals); cultured cells were lysed in PBS containing 1%
Triton X-100 and the same protease inhibitors as above; and a soluble
protein fraction was obtained after collection of the supernatant after
centrifugation. Protein concentration was determined with the Bio-Rad
colorimetric assay system (Bio-Rad Laboratories, Inc.
Hercules, CA). Aliquots of each sample (150 µg protein) were
separated on a 10% SDS-polyacrylamide gel and transferred to
nitrocellulose membrane (Hybond-C-Extra, Amersham Pharmacia Biotech). As a control for the correct protein band, we used
liver tissue from LXR
and LXRß null mice (S. Alberti, G.
Schüster, S. Peterson, and J. Å. Gustafsson,
unpublished data). LXR
proteins were immunochemically
detected using a commercially available antibody (no. SC-1206,
Santa Cruz Biotechnology, Inc.), at a dilution of 2
µg/ml, and signal detection was achieved using ECL chemiluminescence
(Amersham Pharmacia Biotech) according to the
manufacturers instructions.
 |
ACKNOWLEDGMENTS
|
---|
We thank Borgild M. Arntsen and Knut Tomas Dalen for skillful
technical assistance.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Hilde Irene Nebb, Ph.D, Institute for Nutrition Research, Institute of Basic Medical Sciences, University of Oslo, P.O.Box 1046, Blindern, 0316 Oslo, Norway.
1 Both authors contributed equally to these studies and should be
considered jointly as first author. 
Financial support was received from the Norwegian Research Council for
Science and Humanities (NFR), the Norwegian Council for Cancer
Research, the Anders Jahres Foundation for Promotion of Science, Odd
Fellow, Norway, The Norwegian Foundation of Health and Rehabilitation,
the Insulin Fund (Copenhagen, Denmark), the Swedish Medical Research
Council (No. 13X-2819), and Institut Nationale pour la Santé et
la Recherche, and Fondation pour la Recherche Medicale,
France.
Received for publication January 11, 1999.
Revision received October 28, 1999.
Accepted for publication February 1, 2000.
 |
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