Cross-Talk between Fatty Acid and
Cholesterol Metabolism Mediated
by Liver X Receptor-␣
Kari Anne Risan Tobin*, Hilde Hermansen Steineger*,
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
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 LXR␤ (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 5ⴕ-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␣ 5ⴕ-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 cul-
ture 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. (Molecular Endocrinology 14:
741–752, 2000)
INTRODUCTION
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. 1–5). Conversely, fatty acids or their
metabolites are not only reported to be natural ligands
for the different PPARs (reviewed in Refs. 2–5), 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.
0888-8809/00/$3.00/0
Molecular Endocrinology 14(5): 741–752
Copyright © 2000 by The Endocrine Society
Printed in U.S.A.
741
MOL ENDO · 2000
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7), but are also involved in the control of fatty acid and
triglyceride metabolism (8–14).
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 (16–19), including compounds like mevalonate, and naturally occurring oxysterols, such as
22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol,
and 24,25(S)-epoxycholesterol (17–19). Interestingly,
an LXR response element (LXRE) was identified in the
promoter of the rat 7␣-hydroxylase gene, the ratelimiting 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, LXR␤, could not
compensate (20). This suggests that the two isoforms,
LXR␣ and LXR␤, do not have overlapping functions.
They also have differential expression patterns: LXR␤
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␣.
RESULTS
The Effects of Fatty Acids on LXR␣ and LXR␤
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 LXR␤, and we therefore measured the levels of
their respective mRNAs by Northern blot analysis (Ta-
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Table 1. Regulation of LXR␣ and LXR␤ mRNAs in 7800C1
Hepatoma Cells by Fatty Acids
Control
Myristic acid (C 14:0)
Stearic acid (C 18:0)
Oleic acid (C 18:1)
Linolenic acid (C 18:3)
TTA (C 14:S)
LXR␣
LXR␤
1.0
1.42 ⫾ 0.11
1.61 ⫾ 0.24
1.42 ⫾ 0.17
2.05 ⫾ 0.83
3.52 ⫾ 0.05
1.0
0.85 ⫾ 0.05
1.06 ⫾ 0.14
1.16 ⫾ 0.11
0.79 ⫾ 0.13
0.88 ⫾ 0.21
The steady state mRNA levels of these receptors were measured after Northern blot analysis (see Materials and Methods). Total RNA (20 ␮g) from cells treated with 1 mM myristic
acid (C 14:0), 1 mM stearic acid (C 18:0), 1 mM oleic acid (C
18:1), 1 mM linolenic acid (C 18:3), and 50 ␮M TTA was used
in the analysis. All treatments were carried out in duplicates
for 72 h where n ⫽ 6 in a total of three experiments. The
values are presented relative to the control (control ⫽ 1) and
given as the mean ⫾ SEM.
ble 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 LXR␤ was unchanged by all the treatments indicated (Table 1).
Since TTA was the most effective inducer of peroxisomal ␤-oxidation in 7800C1 hepatoma cells (24–
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 timedependent phenotypic changes. We have shown
earlier that these cells express PPAR␣, glucocorticoid, and insulin receptors and possess metabolic
activities such as peroxisomal ␤-oxidation similar to
rat hepatocytes (6, 27, 28). The cells were hence
treated with 50 ␮M 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). LXR␤ 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 Acids Induce LXR␣ Gene Expression
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Fig. 1. Kinetics of LXR mRNA Induction after Treatment of Rat 7800C1 Hepatoma Cells with TTA
A, The steady-state mRNA levels of these receptors were measured after Northern blot analysis (see Materials and Methods)
of total RNA (20 ␮g) from 7800C1 hepatoma cells treated with 50 ␮M TTA for a variable length of time (2–72 h). Relative mRNA
values of LXR␣ (Œ) and LXR␤ (f) are shown. The mRNA values were analyzed in two independent experiments, each carried out
in duplicate (n ⫽ 4). The values are related to control ⫽ 1 and given as the mean ⫾ SEM. B, Northern blot analysis of LXR␣ and
LXR␤ mRNA levels after treatment with TTA. Autoradiograms showing the mRNA level of LXR␣ and LXR␤ in 7800C1 hepatoma
cells treated with 50 ␮M TTA for a varying time period. 18S and 28S rRNA were used to determine the sizes of the mRNA
transcripts.
fatty acid administration, we tested whether TTA treatment affected the stability of LXR␣ or LXR␤ mRNAs.
7800C1 hepatoma cells were treated with TTA (50 ␮M)
for 72 h. The transcriptional inhibitor actinomycin D
(2.5 ␮g/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). LXR␤ 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 ␮M), 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 upregulation of the transcription rate of LXR␤ 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 LXR␤ 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 LXR␤ mRNA were not affected by any of
the fatty acids tested (Fig. 3, A and B).
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 RXR␤ (21).
Taken together, our results show that LXR␣ gene expression is strongly regulated by fatty acids, while
interestingly the isoform LXR␤ 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␣ spe-
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cifically 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 5ⴕ-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 ␮M TTA and 150 ␮M
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).
Fig. 2. Effects of Fatty Acids on LXR mRNA Stability and
Transcription Rates
The mRNA half-life of LXR␣ and LXR␤ after treatment with
TTA. 7800C1 hepatoma cells were incubated with 50 ␮M TTA
for 3 days. Actinomycin D (AD) (2.5 ␮g/ml) was then added
and the cells were harvested at different time points up to
12 h. The resulting Northern filters were hybridized to 32Plabeled probe for the indicated receptors and subjected to
autoradiography. Suitable saturated autoradiograms were
scanned for semiquantitative assessment of the mRNA for
each receptor. mRNA level relative to control (C) at each time
point (control ⫽ 1) was plotted in a time curve, and half-lives
were estimated by extrapolation in the linear part of the
mRNA time curve. Panel A shows a representative time curve
from one of the experiments that were repeated three times.
Panel B shows the calculated half-lives for the LXR␣ (u) and
LXR␤ (s) transcripts. C, Nuclear run-on of LXR␣ and LXR␤
transcription rates after treatment with TTA. Transcription
run-on assays were performed with nuclei isolated from
7800C1 hepatoma cells treated for 2, 4, and 6 h with TTA (50
␮M). The figure shows the optical density measured at different time points for each of the above mentioned receptors.
Relative mRNA values of LXR␣ (Œ) and LXR␤ (f) are shown.
Also included in the assay were the ribosomal protein L27 (䡺)
acting as a control, and the vectors that the different receptors were cloned into: pBluescript (SK⫹) (‚) and pGEM-T
vector (E). The figure shows the results from one experiment,
which was repeated once with similar results.
Fatty Acids Induce LXR␣ Gene Expression
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Fig. 3. Effects of Unsaturated Fatty Acids on LXR␣ and LXR␤ mRNA Levels in Cultured Rat Hepatocytes after Treatment for 24 h
A, Autoradiograms showing the mRNA level of LXR␣ and LXR␤ in cultured primary rat hepatocytes treated with 1 mM oleic acid
(C18:1), 1 mM linolenic acid (C18:3), and 50 ␮M 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␣ (u) and LXR␤ (s) 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 ␮M 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.
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).
DISCUSSION
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
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Fig. 4. Effects of Unsaturated Fatty Acids on LXR␣ Protein Levels in Cultured Rat Hepatocytes
LXR␣ protein level in tissues from wild-type and knockout mice (LXR␣⫺/⫺ and LXR␤⫺/⫺) and cultured hepatocytes was studied.
Total protein fraction was prepared and subjected to immunoblotting as described in Materials and Methods. Samples of protein
(150 ␮g) were subjected to electrophoresis on denaturing SDS-polyacrylamide gels and transferred to nitrocellulose membrane.
A, To verify the correct protein band, immunoblots using protein lysates from livers of wild-type and LXR␣ knockout mice were
prepared. The polyclonal antibody against LXR␣ recognized a band at 51 kDa which is in agreement with the published molecular
mass. The immunocomplexes were visualized by ECL. B, Kinetics of the LXR␣ protein level in cultured hepatocytes treated with
1 mM linolenic acid for up to 48 h. C, Effect of 1 mM oleic acid (18:1) and linolenic acid (18:3). D, 50 ␮M TTA and 100 ␮M Wy 14.643
on LXR␣ protein level in cultured hepatocytes stimulated for 24 h.
gene expression by lipids belong to the PPAR and
SREBP families of transcription factors (reviewed in
Refs. 1–7). 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.
1–7). The nuclear receptor LXR␣ was recently shown
to be a transcriptional modulator capable of responding to changes in levels of cholesterol metab-
Fatty Acids Induce LXR␣ Gene Expression
747
Fig. 5. The 5⬘-Flanking Region of the mLXR␣ Gene Is Regulated by PPAR␣
A, A construct containing 1500 bp of the 5⬘-flanking region of the mLXR␣ gene in front of a luciferase reporter (pLXR␣(⫺1500/
⫹1800)LUC) (Materials and Methods) was cotransfected with 0.4 ␮g of an expression plasmid of RXR␣ (pCMV-RXR␣) with or
without 0.4 ␮g of an expression plasmid encoding PPAR␣ (pSG5-PPAR␣) into COS-1 cells. The cells were stimulated with
increasing concentrations of TTA or Wy 14.643 and harvested after 72 h. Luciferase activity was measured and normalized against
␤-galactosidase activity. The values are presented relative to unstimulated reporter gene activity cotransfected with RXR␣
(control ⫽ 1) and given as the mean ⫾ SEM from three independent experiments. B, Deletion constructs of the 5⬘-flanking region
of LXR␣ were transfected into COS-1 cells in the same way as above and stimulated with 50 ␮M TTA. All wells received 0.4 ␮g
RXR␣ expression plasmid, and 0.4 ␮g PPAR␣ expression plasmid where indicated. Fold induction was calculated relative to each
deletion construct. Each point represents the mean ⫾ SEM from at least two independent experiments. C, Potential PPREs located
in the 5⬘-flank of the mLXR␣ gene. Computer homology search of the 5⬘-flanking region of LXR␣ (the pLXR␣(⫺1500)LUCconstruct) identified five potential fatty acid response elements (PPRE1–PPRE5). The PPREs are located between ⫺1144 to ⫹
150 relative to the transcriptional start site, and the sequences of these elements are listed below. Also included below is the
consensus PPRE and PPREs found in a selected group of genes important in lipid metabolism (46–49).
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Fig. 6. Effect of PUFAs and PPAR␣ Activators on the LXR␣ mRNA and Protein Levels in Vivo
A, High-fat diet (15% Soy oil, PUFA) was given to rats for 48 h. The effects on liver LXR␣ mRNA level and protein level were
examined (Materials and Methods). B, Rats were given different PPAR␣ activators by gastric intubation once a day for 3 days.The
effects on liver LXR␣ mRNA level and protein level were examined (Materials and Methods). 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). Control animals received only CMC.
olites, suggesting that it could be a pivotal cholesterol sensor (16, 18–20). 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 PPRElike 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
Fig. 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.
interaction. In general, when the organism senses
low cholesterol level, SREBP is activated and upregulates 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).
Fatty Acids Induce LXR␣ Gene Expression
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
␤-oxidation or stored as an energy reserve in the
adipocytes (reviewed in Refs. 1–7). 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 ␤-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.
749
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.
MATERIALS AND METHODS
Materials
Ham’s 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 h–2000 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 ⫻ 20-mm culture
dishes (Greiner, Kremünster, Austria) and plated at 2–4 ⫻ 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
MOL ENDO · 2000
750
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 ␮g/ml),
fungizone (2.5 ␮g/ml), and anti-pleuropneumonia-like organisms (50 ␮g/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 ⫻
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 ␮g test plasmid
and 5 ␮g ␤-galactosidase plasmid as internal control, and 0.4
␮g 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 ␤-galactosidase activity measured by incubating 10 ␮l extract with 0.28 mg o-nitrophenyl-␤-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 ␮M); 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 acidfree BSA to the cells. The concentration of TTA (50 ␮M) 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-BlueExclusion 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 LXR␤ (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).
Vol 14 No. 5
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 2–6 ⫻ 108 cpm/␮g 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
(5–30 ␮g 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 LXR␤ (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 ␮M) 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 ␮g/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 ␮g 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 LXR␤ 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 ␮g/ml, and signal
detection was achieved using ECL chemiluminescence (Amersham Pharmacia Biotech) according to the manufacturer’s
instructions.
Acknowledgments
We thank Borgild M. Arntsen and Knut Tomas Dalen for
skillful technical assistance.
Fatty Acids Induce LXR␣ Gene Expression
Received January 11, 1999. Re-revision received October
28, 1999. Accepted February 1, 2000.
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. E-mail: [email protected].
* 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.
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