Cross-Talk between Fatty Acid and Cholesterol Metabolism Mediated by Liver X Receptor-{alpha}

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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
LXR{alpha} (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{alpha} 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{alpha} (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{alpha} gene account for the major part of the induction of LXR{alpha} 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{alpha} gene, show a peroxisome proliferator-activated receptor-{alpha}-dependent increase in luciferase activity upon treatment with tetradecylthioacetic acid and the synthetic peroxisome proliferator-activated receptor-{alpha} activator, Wy 14.643, suggesting that the mLXR{alpha} 5'-flanking region contains the necessary sequence elements for fatty acid responsiveness. In addition, in vivo LXR{alpha} expression was induced by fatty acids, consistent with the in vitro cell culture data. These observations demonstrate that LXR{alpha} expression is controlled by fatty acid signaling pathways and suggest an important cross-talk between fatty acid and cholesterol regulation of lipid metabolism.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha} (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 {alpha} [LXR{alpha}, 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{alpha}-hydroxylase gene, the rate-limiting enzyme in the conversion of cholesterol into bile acids (16, 19). Further evidence supporting an important role of LXR{alpha} in lipid homeostasis was provided by the loss of capacity to regulate catabolism of dietary cholesterol in mice in which the LXR{alpha} 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{alpha} and LXRß, do not have overlapping functions. They also have differential expression patterns: LXRß is ubiquitously expressed, whereas LXR{alpha} 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{alpha}, we were interested in determining whether a similar regulation by metabolites from lipid metabolism occurs for LXR{alpha}. We therefore analyzed whether LXR{alpha} expression is modified by fatty acids in the liver. Our results suggest that LXR{alpha} 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{alpha}.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Effects of Fatty Acids on LXR{alpha} 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{alpha} and RXR{alpha} mRNA in hepatoma cells and cultured hepatocytes (6, 21, 22). LXR{alpha} has been shown to have the same tissue distribution as PPAR{alpha} and RXR{alpha} (1, 23). In addition, LXR{alpha} 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{alpha} and LXRß, and we therefore measured the levels of their respective mRNAs by Northern blot analysis (Table 1Go and Fig. 1Go, 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 1Go). With nonmodified fatty acids, only slight inductions of the LXR{alpha} mRNA level were observed (for instance, 2-fold induction with C18:3). However, TTA resulted in a 3.5-fold induction of LXR{alpha} mRNA. The mRNA level of LXRß was unchanged by all the treatments indicated (Table 1Go).


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Table 1. Regulation of LXR{alpha} and LXRß mRNAs in 7800C1 Hepatoma Cells by Fatty Acids

 


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Figure 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 µg) from 7800C1 hepatoma cells treated with 50 µM TTA for a variable length of time (2–72 h). Relative mRNA values of LXR{alpha} ({blacktriangleup}) and LXRß ({blacksquare}) 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{alpha} and LXRß mRNA levels after treatment with TTA. Autoradiograms showing the mRNA level of LXR{alpha} and LXRß in 7800C1 hepatoma cells treated with 50 µM TTA for a varying time period. 18S and 28S rRNA were used to determine the sizes of the mRNA transcripts.

 
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{alpha} mRNA level in rat hepatoma cells, this fatty acid analog was further used to investigate the kinetics of the LXR{alpha} 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{alpha}, 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. 1Go, 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. 1Go, A and B). A decline in the LXR{alpha} mRNA level was observed after 72 h (Fig. 1Go, A and 1B). LXRß expression was not significantly affected by these treatments (Fig. 1Go, A and B).

Fatty Acids Increase LXR{alpha} mRNA Stability and Transcription Rate
To further define the mechanism underlying the elevated LXR{alpha} steady state mRNA level observed after fatty acid administration, we tested whether TTA treatment affected the stability of LXR{alpha} 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. 2AGo). Actinomycin D inhibited the LXR{alpha} mRNA synthesis and prevented further induction by TTA. TTA led to an increase in the half-lives of LXR{alpha} transcripts as compared with the control (5.8 and 4.0 h, respectively; Fig. 2BGo). LXRß mRNA stability remained constant under all conditions tested (Fig. 2Go, A and B). These results indicate that the increase in the steady state levels of mRNA for LXR{alpha} in 7800C1 hepatoma cells after TTA treatment is at least partially due to a stabilization of the LXR{alpha} mRNA.



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Figure 2. Effects of Fatty Acids on LXR mRNA Stability and Transcription Rates

The mRNA half-life of LXR{alpha} and LXRß after treatment with TTA. 7800C1 hepatoma cells were incubated with 50 µM TTA for 3 days. Actinomycin D (AD) (2.5 µg/ml) was then added and the cells were harvested at different time points up to 12 h. The resulting Northern filters were hybridized to 32P-labeled 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{alpha} () and LXRß () transcripts. C, Nuclear run-on of LXR{alpha} and LXRß 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 µM). The figure shows the optical density measured at different time points for each of the above mentioned receptors. Relative mRNA values of LXR{alpha} ({blacktriangleup}) and LXRß ({blacksquare}) are shown. Also included in the assay were the ribosomal protein L27 ({square}) acting as a control, and the vectors that the different receptors were cloned into: pBluescript (SK+) ({triangleup}) and pGEM-T vector ({circ}). The figure shows the results from one experiment, which was repeated once with similar results.

 
The stabilization of LXR{alpha} mRNA is, however, insufficient to explain the marked increase in the LXR{alpha} 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 2CGo 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{alpha} 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. 2CGo). The empty vector backbone for the LXR{alpha} and LXRß cDNAs were used as negative controls. These results indicate that the up-regulated level of LXR{alpha} 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{alpha} mRNA Levels in Primary Rat Hepatocyte Cultures
To study how fatty acids induce the LXR{alpha} 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. 3Go, 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{alpha} mRNA levels after 24 h relative to untreated cells (Fig. 3Go, A and B). Again, the levels of LXRß mRNA were not affected by any of the fatty acids tested (Fig. 3Go, A and B).



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Figure 3. Effects of Unsaturated Fatty Acids on LXR{alpha} and LXRß mRNA Levels in Cultured Rat Hepatocytes after Treatment for 24 h

A, Autoradiograms showing the mRNA level of LXR{alpha} 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{alpha} () 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.

 
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 {alpha} (RAR{alpha}) (data not shown) and the ribosomal protein L27 (Fig. 3AGo), as well as shown in earlier studies for RXRß (21). Taken together, our results show that LXR{alpha} 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{alpha} Protein Levels in Primary Rat Hepatocyte Cultures
After studies of the fatty acid-induced mRNA expression of LXR{alpha} in primary rat hepatocytes, we studied whether the induction could also be seen at protein level. LXR{alpha} protein was monitored in liver protein extracts by immunoblotting. Antisera against LXR{alpha} specifically recognized a band at 51 kDa, which is in agreement with its calculated mol wt (Fig. 4AGo)(15). LXR{alpha} 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. 4Go B). The monounsaturated fatty acid oleic acid (18:1) induced LXR{alpha} protein level 2.3-fold and linolenic acid 1.8-fold in cultured hepatocytes 24 h after stimulation (Fig. 4CGo). These results are in concordance with the fatty acid regulation of LXR{alpha} mRNA shown in Fig. 3Go, A and B. Treatment of primary hepatocytes with TTA and Wy 14.643, a synthetic PPAR{alpha} activator, gave only minor induction of LXR{alpha} protein.



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Figure 4. Effects of Unsaturated Fatty Acids on LXR{alpha} Protein Levels in Cultured Rat Hepatocytes

LXR{alpha} protein level in tissues from wild-type and knockout mice (LXR{alpha}-/- and LXRß-/-) and cultured hepatocytes was studied. Total protein fraction was prepared and subjected to immunoblotting as described in Materials and Methods. Samples of protein (150 µg) 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{alpha} knockout mice were prepared. The polyclonal antibody against LXR{alpha} 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{alpha} 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 µM TTA and 100 µM Wy 14.643 on LXR{alpha} protein level in cultured hepatocytes stimulated for 24 h.

 
The 5'-Flanking Region of the Mouse (m)LXR{alpha} Gene Confers Responsiveness to Fatty Acids
To examine the upstream region of the mLXR{alpha} gene for sequences that might mediate the transcriptional effect of fatty acids on LXR{alpha} gene expression, a luciferase reporter gene construct containing part of the 5'-flanking region of LXR{alpha} gene was used. About 1,500 bp of the LXR{alpha} 5'-flanking sequence (LXR{alpha}(-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{alpha} (pCMV-RXR{alpha}). Stimulation with increasing doses of either TTA or the specific PPAR{alpha} activator, Wy 14.643, gave only minor effects on luciferase activity (Fig. 5AGo). Cotransfection of PPAR{alpha} expression plasmid (pSG5-PPAR{alpha}) in addition to RXR{alpha} expression vector without any stimulation gave 2.6-fold induction, indicating the presence of endogenous ligands for PPAR{alpha} in COS-1 cells. However, cotransfection of PPAR{alpha} 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. 5AGo). Concentrations above 100 µM TTA and 150 µM Wy 14.643 was toxic to the cells. These observations indicate that PPAR{alpha} 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).



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Figure 5. The 5'-Flanking Region of the mLXR{alpha} Gene Is Regulated by PPAR{alpha}

A, A construct containing 1500 bp of the 5'-flanking region of the mLXR{alpha} gene in front of a luciferase reporter (pLXR{alpha}(-1500/+1800)LUC) (Materials and Methods) was cotransfected with 0.4 µg of an expression plasmid of RXR{alpha} (pCMV-RXR{alpha}) with or without 0.4 µg of an expression plasmid encoding PPAR{alpha} (pSG5-PPAR{alpha}) 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 ß-galactosidase activity. The values are presented relative to unstimulated reporter gene activity cotransfected with RXR{alpha} (control = 1) and given as the mean ± SEM from three independent experiments. B, Deletion constructs of the 5'-flanking region of LXR{alpha} were transfected into COS-1 cells in the same way as above and stimulated with 50 µM TTA. All wells received 0.4 µg RXR{alpha} expression plasmid, and 0.4 µg PPAR{alpha} 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{alpha} gene. Computer homology search of the 5'-flanking region of LXR{alpha} (the pLXR{alpha}(-1500)LUC-construct) 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 47 48 49 ).

 
Next, different 5'-deletion constructs of the LXR{alpha} 5'-flanking region were transfected into COS-1 cells. The PPAR{alpha}-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{alpha}(-700/+1800)LUC] and 5-fold [LXR{alpha}(-100/+1800)LUC], and finally down to 2.5-fold for a construct [LXR{alpha}(-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{alpha} gene (Fig. 5BGo).

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{alpha} gene indicates the presence of several sequence elements with a good homology to the consensus PPRE (Fig. 5CGo).

These elements need to be further examined in future studies.

Effect of Polyunsaturated Fatty Acids (PUFAs) and PPAR{alpha} Agonists on the LXR{alpha} mRNA and Protein Levels in Vivo
To establish the relevance of these in vitro observations, we examined the effect of PUFAs on liver LXR{alpha} 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{alpha} mRNA level 3-fold (Fig. 6AGo, upper panel), whereas semiquantitative immunoblot analysis of LXR{alpha} protein from rat liver showed a 3.3-fold increase after feeding with the PUFA diet (Fig. 6AGo, lower panel).



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Figure 6. Effect of PUFAs and PPAR{alpha} Activators on the LXR{alpha} 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{alpha} mRNA level and protein level were examined (Materials and Methods). B, Rats were given different PPAR{alpha} activators by gastric intubation once a day for 3 days.The effects on liver LXR{alpha} mRNA level and protein level were examined (Materials and Methods). The PPAR{alpha} 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.

 
Rats were then given TTA, Wy 14.643, or linolenic acid by gastric intubation for 3 days (Materials and Methods). Liver LXR{alpha} mRNA in animals given different PPAR{alpha} activators was induced approximately 2-fold for TTA and Wy 14.643, but to a lower degree by linolenic acid (Fig. 6BGo, upper panel), whereas LXR{alpha} protein expression was induced 3-fold by the potent PPAR{alpha}-agonist Wy 14.643, but also by TTA and linolenic acid (1.8-fold and 2.2-fold, respectively)(Fig. 6BGo, lower panel).

Taken together, feeding rats a diet rich in unsaturated fatty acids, or other PPAR{alpha} activators, results in an induction in LXR{alpha} mRNA and protein level. These data are concordant with the observed changes in LXR{alpha} mRNA and protein levels after fatty acid treatment of rat hepatocyte and hepatoma cultures (Figs. 1Go, 3Go, and 4Go).

Effects of Fasting-Refeeding on LXR{alpha} mRNA Steady State Levels
Finally, we investigated whether the LXR{alpha} 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{alpha} mRNA steady state level by approximately 3-fold (Fig. 7Go). 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{alpha} 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{alpha} mRNA by Northern blotting (see Materials and Methods). The figure represents data from one experiment and has been repeated with similar results.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha} 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{alpha} expression is induced by fatty acids. This effect seems to be the consequence of a direct induction of LXR{alpha} gene transcription, mediated through putative fatty acid response elements located in the proximal LXR{alpha} 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{alpha} 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{alpha} gene expression observed. Recently it was suggested that PPAR{alpha} has a role in the transcriptional response to fasting, since fasting induces several PPAR{alpha} target genes encoding enzymes involved in the fatty acid oxidative pathway, an effect abolished in PPAR{alpha}-/- mice (30, 31).

The transcription factors involved in mediating this effect of fatty acids on LXR{alpha} 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{alpha} 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{gamma} ligands, SREBP was recently shown to directly induce transcription of the PPAR{gamma} gene (11, 32). Certain fatty acids, the end products of the enzymatic pathways SREBP and PPAR{gamma} are involved in, potentiate the SREBP-regulated gene transcription (33, 34).

In addition, LXR{alpha} 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{alpha} 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{alpha} by fatty acids, primarily in liver and muscle, enhances energy production through its stimulating effects on the ß-oxidation pathways, whereas PPAR{gamma} 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{alpha} gene expression (cross-regulatory loop). In addition, cholesterol in food provides potential ligands and activators of LXR{alpha} receptor. Through induction of LXR{alpha} levels (via fatty acids) and through the enhanced supply of its cholesterol-derived ligands, the LXR{alpha}-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{alpha} expression by fatty acids and suggest that this allows cross-talk between gene regulation by fatty acids and cholesterol, respectively.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 [{alpha}-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{alpha} 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{alpha} activators by gastric intubation once a day for 3 days. The PPAR{alpha} 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 2–4 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{alpha} or pSG5-PPAR{alpha} 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{alpha} (15), rat LXRß (41), and glyceraldehydes-3-phosphate dehydrogenase (42) were used as probes in Northern hybridizations. Murine LXR{alpha} 5'-flanking reporter construct was made by subcloning the 5'-upstream regulatory sequence between -1500 bp and +1800 of the mLXR{alpha} gene into the pGL3-basic luciferase reporter plasmid to yield the pLXR{alpha}(-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{alpha} 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). [{alpha}-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 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 (5–30 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{alpha} [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{alpha} and LXRß null mice (S. Alberti, G. Schüster, S. Peterson, and J. Å. Gustafsson, unpublished data). LXR{alpha} 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 manufacturer’s 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. Back

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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 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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