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Endocrinology Vol. 146, No. 12 5380-5387
Copyright © 2005 by The Endocrine Society

Hepatic Peroxisomal Fatty Acid ß-Oxidation Is Regulated by Liver X Receptor {alpha}

Tonghuan Hu, Patricia Foxworthy, Angela Siesky, James V. Ficorilli, Hong Gao, Shuyu Li, Michael Christe, Timothy Ryan, Guoqing Cao, Patrick Eacho, M. Dodson Michael and Laura F. Michael

Lilly Research Laboratories, Departments of Cardiovascular Research (T.H., P.F., J.V.F., M.C., G.C., P.E., L.F.M.), Endocrinology (A.S., M.D.M.), Toxicology (H.G., T.R.), Integrative Biology (S.L.), Eli Lilly & Co., Indianapolis, Indiana 46285

Address all correspondence and requests for reprints to: Dr. Laura F. Michael, Eli Lilly & Co., Cardiovascular Research, DC 0520, Indianapolis, Indiana 46285. E-mail: laura_michael{at}lilly.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Peroxisomes are the exclusive site for the ß-oxidation of very-long-chain fatty acids of more than 20 carbons in length (VLCFAs). Although the bulk of dietary long-chain fatty acids are oxidized in the mitochondria, VLCFAs cannot be catabolized in mitochondria and must be shortened first by peroxisomal ß-oxidation. The regulation of peroxisomal, mitochondrial, and microsomal fatty acid oxidation systems in liver is mediated principally by peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}). In this study we provide evidence that the liver X receptor (LXR) regulates the expression of the genetic program for peroxisomal ß-oxidation in liver. The genes encoding the three enzymes of the classic peroxisomal ß-oxidation cycle, acyl-coenzyme A (acyl-CoA) oxidase, enoyl-CoA hydratase/L-3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase, are activated by the LXR ligand, T0901317. Accordingly, administration of T0901317 in mice promoted a dose-dependent and greater than 2-fold increase in the rate of peroxisomal ß-oxidation in the liver. The LXR effect is independent of PPAR{alpha}, because T0901317-induced peroxisomal ß-oxidation in the liver of PPAR{alpha}-null mice. Interestingly, T0901317-induced peroxisomal ß-oxidation is dependent on the LXR{alpha} isoform, but not the LXRß isoform. We propose that induction of peroxisomal ß-oxidation by LXR agonists may serve as a counterregulatory mechanism for responding to the hypertriglyceridemia and liver steatosis that is promoted by potent LXR agonists in vivo; however, additional studies are warranted.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FREE FATTY ACIDS derived mainly from adipose tissue are delivered to liver for catabolism via mitochondrial or peroxisomal ß-oxidation. Fatty acid oxidation occurs in three major steps: activation of fatty acids through formation of a high energy thioester bond with cystolic free coenzyme A (CoA) generating fatty acyl-CoA, removal of C2 acetyl-CoA units by ß-oxidation, and ATP synthesis from entry of the liberated acetyl-CoA moieties into the citric acid cycle (reviewed in Ref.1). Mitochondria as well as peroxisomes oxidize fatty acids via ß-oxidation, with long-chain and very-long-chain fatty acids (VLCFAs; C24:0 and C26:0) being oxidized preferentially by peroxisomes (reviewed in Ref.2). Complete oxidation of fatty acids to carbon dioxide and water occurs only in mitochondria, because peroxisomes lack enzymes of the citric acid cycle.

Although the bulk of dietary long chain fatty acids are oxidized in the mitochondria, VLCFAs cannot be catabolized in mitochondria and must be shortened first by peroxisomal ß-oxidation. In the classical peroxisomal ß-oxidation pathway, dehydrogenation of acyl-CoA esters to trans-2-enoyl-CoAs is catalyzed by fatty acyl-CoA oxidase (Acox), and the second and third reactions, hydration and dehydrogenation of enoyl-CoA esters to 3-ketoacyl-CoA (Acaa1), are catalyzed by a multifunctional enzyme, enoyl-CoA hydratase/liter-3-hydroxyacyl-CoA dehydrogenase (Ehhadh). Finally, 3-oxoacyl-CoA thiolase cleaves 3-oxoacyl-CoAs to acetyl-CoA. Thus, the original acyl-CoA substrate that is two carbons shorter may reenter the ß-oxidation cycle, and the newly generated acetyl-CoA moieties enter the citric acid cycle (reviewed in Ref.2).

The genes encoding the classical peroxisomal ß-oxidation pathway enzymes are markedly induced in response to structurally diverse agents known as peroxisome proliferators (reviewed in Ref.3). Mechanistically, peroxisome proliferators bind and activate the peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}), thereby promoting PPAR{alpha}-mediated target gene transcription. Indeed, the fibrate class of peroxisome proliferator molecules is clinically successful in treating hypertriglyceridemia and mixed hyperlipidemia, presumably through PPAR{alpha}-dependent induction of genes that control fatty acid ß-oxidation (4).

The liver X receptors (LXRs) also play pivotal roles in regulating the expression of the genes involved in fatty acid metabolism. LXR agonists induce the transcription of genes involved in fatty acid synthesis, including sterol response element-binding protein 1c (SREBP1c), the master regulator of fatty acid synthesis (5, 6, 7). Genes encoding the enzymes involved in fatty acid synthesis, including fatty acid synthase (FASN) and stearoyl-CoA desaturase 1, are regulated either directly or indirectly by LXR (6, 8). In addition to increasing expression of the lipogenic gene program, LXR controls the expression of genes involved in catabolism of cholesterol to bile acids, in regulation of several genes important for reverse cholesterol transport from peripheral tissues, in high-density lipoprotein accumulation, and in cholesterol excretion into bile or the intestinal lumen (9, 10, 11, 12, 13, 14, 15, 16). In this study we provide evidence that LXR also regulates the expression of the genetic program for peroxisomal ß-oxidation in liver independently of PPAR{alpha}. Thus, LXR agonists may induce peroxisomal ß-oxidation as a counterregulatory response to the hypertriglyceridemia and liver steatosis that are also promoted by potent LXR agonists in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal care and treatments
Male 129/Sv and PPAR{alpha}-null mice, 129s4 SvJae-Ppara<tm1Gonz>, were purchased from The Jackson Laboratory (Bar Harbor, ME), and C57BL/6 mice were purchased from Harlan Laboratories (Indianapolis, IN). Animals were acclimated for 1 wk before study initiation. Mice were housed five per cage in polycarbonate cages with filter tops. Animals were maintained on a 12-h light, 12-h dark cycle (lights on at 0600 h) at 21 C. All animals received deionized water and the 5008 Diet (PMI Nutrition International, Brentwood, MO) ad libitum. All animals were maintained in accordance with the institutional animal use and care committee of Eli Lilly & Co. and the National Institutes of Health Guide for the Use and Care of Laboratory Animals. Antisense oligonucleotides (ASOs) were prepared in normal saline, and the solution was sterilized through a 0.2-µm pore size filter. Animals were treated with ASO solutions or vehicle (saline) twice per week (separated by 3.5 d) via sc injection. T0901317 was administered once daily in peanut oil vehicle by oral gavage at the indicated doses. Fenofibrate was administered once daily in 1% (wt/vol) carboxymethylcellulose and 0.25% Tween 80 by oral gavage.

Design and identification of lead LXR ASO inhibitors
ASOs targeting mouse LXR{alpha} and LXRß sequences were designed as 20-base full-phosphorothioate chimeric 2'-O-(2-methoxy)-ethyl modified ASOs. These molecules represent a class of 20-base chimeric ASOs in which a ribonuclease H-sensitive stretch of 10 2'-deoxy residues is flanked on both sides with a stretch of five 2'-O-(2-methoxy)-ethyl modifications. These modifications increase mRNA binding affinity and confer nuclease resistance. Such a chimeric design provides an attractive pharmacological and toxicological profile while maintaining the highly efficient ribonuclease H terminating mechanism (17). Mouse LXR{alpha} ASO (5'-TGCCTCCCTGGTCTCCTGCA-3') and mouse LXRß ASO (5'-TTCCGAATCTGCTCCTCAGA-3') were identified as potent ASOs. The control ASO (sequence 5'-CCTTCCCTGAAGGTTCCTCC-3') used in these studies is of the same chemistry, design, and length as the LXR ASOs, but it lacks perfect complementarity to any rodent gene in public databases.

Oligonucleotide treatments of cells
To assess and optimize LXR mRNA reduction in vitro, primary mouse hepatocytes were plated at 10,000 cells/well in a 96-well plate and were grown in William’s E medium, 10% fetal bovine serum, 1% nonesterified essential fatty acids, 1% L-glutamine, and 50 mM HEPES containing antibiotic/antimycotic. The cells were transfected with varying concentrations of LXR ASOs using Lipofectin (Invitrogen Life Technologies, Inc., Carlsbad, CA) solution for 4 h. Target reduction was determined 20 h after transfection by real-time PCR analysis.

Microarray analysis of liver gene expression
Total RNA was isolated from approximately 100 mg mouse liver tissue using TRIzol methodology (Invitrogen Life Technologies, Inc.). RNA was additionally purified using RNeasy columns (Qiagen, Valencia, CA). Equal amounts of RNA from each animal within treatment groups were pooled, and samples were prepared for GeneChip analysis according to the Affymetrix eukaryotic expression sample protocol revision 1 (Affymetrix, Santa Clara, CA). Samples were then hybridized to MG-U74Av2 oligonucleotide arrays. Data analysis and mining were performed using Affymetrix Microarray suite (MAS 4.0) and data mining tool (DMT 2.0) software.

Real-time PCR analysis of target gene expression
Real-time quantitative PCR was performed using the 5'-fluorogenic nuclease assay and ABI 7900 PRISM (Applied Biosystems, Foster City, CA) to determine the relative abundance of assayed mRNAs. Samples were normalized using Ribogreen (Molecular Probes, Eugene, OR) or by determining the relative abundance compared with 36B4 mRNA. The 5' terminus of fluorogenic probes was labeled with 6-carboxyfluorescein, and the 3' terminus contained the quenching dye 6-carboxytetramethylrhodamine. Sequences were as follows: mouse 36B4 forward primer, 5'-GGCCCGAGAAGACCTCCTT-3'; mouse 36B4 reverse primer, 5'-TCAATGGTGCCTCTGGAGATT-3'; mouse 36B4 TaqMan probe, 5'-CCAGGCTTTGGGCATCACCACG-3'; mouse SREBP1 forward primer, 5'-TTGGCCACAGTACCTTTGGTT-3'; mouse SREBP1 reverse primer, 5'-CTGAGCCTAGGGCCTTGCT-3'; mouse SREBP1 TaqMan probe, 5'-CATCCACCGACTCGCAGCTGG-3'; mouse FASN forward primer, 5'-GCCTGGACTCGCTCATGG-3'; mouse FASN reverse primer, 5'-TGAAGTTTCCGCAGCGTG-3'; and mouse FASN TaqMan probe, 5'-CGTCAGATCCTGGAACGAGAACACGATC-3'. TaqMan primer-probe sets for the following genes were obtained from TaqMan Assays-on-Demand Gene Expression Products (Applied Biosystems): mouse Acox (Mm00443579_ml), mouse Ehhadh (Mm00470091_sl), mouse Acaa1 (Mm00728460_ml), mouse CPT1a (Mm00550438_m1), mouse LCAD (Mm005599660_m1), mouse MCAD (Mm00431611_m1), mouse uncoupling protein 2 (Mm00495907_g1), and mouse Cyp7A1 (Mm00484152_m1). PCRs were run in quadruplicate 10-µl reactions in 384-well plates that contained Universal Master Mix (Applied Biosystems), 2 pmol of each forward and reverse primer, 1.5 pmol probe, and cDNA. Statistical analysis using one-way ANOVA, followed by comparison with vehicle or T0901317 treatment, followed by Dunnett’s method, was performed as described in each figure legend.

Measurement of liver peroxisomal ß-oxidation
Peroxisome-mediated fatty acid ß-oxidation activity was measured as described previously (18). Briefly, 100 mg liver was homogenized in 9 vol cold 0.25 M sucrose with silicon beads (BIO 101, Carlsbad, CA). Samples were centrifuged at 600 x g for 10 min, and the upper lipid layer was aspirated. Triton X-100 (50 µl of a 10% solution) was added to each 450 µl sample supernatant. The samples were then assayed for peroxisomal ß-oxidation in the presence of potassium cyanide, which inhibits mitochondrial ß-oxidation. The oxidation of palmitoyl-CoA was quantified spectrophotometrically by measuring the reduction of nicotinamide adenine dinucleotide-positive at 340 nm. The rate of nicotinamide adenine dinucleotide-positive reduction is directly related to the rate of fatty acid oxidation. Statistical analysis using one-way ANOVA, followed by comparison with vehicle or T0901317 treatment, followed by Dunnett’s method, was performed as described in each figure legend.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LXR regulates genes of the ß-oxidation pathway
Previous studies have shown that PPAR{alpha} agonists activate transcription of genes in the classical peroxisomal fatty acid ß-oxidation pathway, such as Acox, Ehhadh, and Acaa1 (3), resulting in elevated fatty acid oxidation in liver. PPAR{alpha} agonists also are known to induce the expression of LXR (19, 20, 21). Therefore, we were intrigued to find that the genes of the peroxisomal ß-oxidation pathway displayed increased mRNA levels in a microarray analysis of liver samples from mice treated with the potent LXR agonist, T0901317. Specifically, the expression of the three peroxisomal ß-oxidation genes, Acox, Ehhhadh, and Acaa1, was elevated (Table 1Go).


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  		TABLE 1. Peroxisomal ß-oxidation genes

 
To confirm the microarray observations and to determine the dose-responsive nature of the target gene expression, we evaluated the mRNA levels of these genes from livers of C57BL/6 mice treated for 1 wk with doses of T0901317 ranging from 1–30 mg/kg. Quantitative real-time (Q-RT) PCR analysis indicated that transcription of all three genes responded positively in a dose-responsive manner, and among them, Ehhadh demonstrated the largest transcriptional response (Fig. 1AGo). To functionally evaluate the consequence of increased expression of the peroxisomal ß-oxidation genetic program, we examined the ability of T0901317 to regulate peroxisomally mediated ß-oxidation of palmitoyl-CoA in the same liver samples. The LXR agonist induced peroxisomal ß-oxidation in a dose-dependent manner, with a peak induction of 2-fold at the 30 mg/kg dose (Fig. 1BGo).



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  		FIG. 1. LXR agonist induction of the ß-oxidation pathway is dose dependent. A and C, Quantitative measurement of selected target gene transcripts by Q-RT PCR performed using RNA isolated from liver tissue of C57BL/6 mice treated with T0901317 at the indicated doses for 1 wk (n = 5/group). Data are normalized to the 36B4 gene and are presented as the mean ± SEM. Statistical analysis using one-way ANOVA, followed by comparison with vehicle by Dunnett’s method, were performed. *, P ≤ 0.05. B, Measurement of peroxisome-mediated ß-oxidation of lipids was performed using liver tissue of C57BL/6 mice treated with T0901317 at the indicated doses for 1 wk. Data are presented as the mean ± SEM. Statistical analysis using one-way ANOVA, followed by comparison with vehicle by Dunnett’s test, were performed. *, P ≤ 0.05.

 
We also explored potential regulation of key genes involved in mitochondrial ß-oxidation by LXR. Carnitine palmitoyl transferase, which mediates translocation of acyl-CoA esters across the inner mitochondrial membrane, and the dehydrogenases, medium-chain acyl-CoA dehydrogenase and long-chain acyl-CoA dehydrogenase play roles in controlling mitochondrial ß-oxidation flux. In contrast to the significant elevation in genes expressing peroxisomal ß-oxidation enzymes, the carnitine palmitoyl transferase gene was not elevated by T0901317, and the long-chain acyl-CoA dehydrogenase gene was elevated only at the high dose of T0901317 (Fig. 1CGo). However, the response of the medium-chain acyl-CoA dehydrogenase gene to T0901317 was similar to that of Acox and Acaa1 gene inductions. We also found that the expression of the ubiquitous mitochondrial transporter, uncoupling protein 2, was increased dose-dependently by T0901317. Because the genes involved and the rates of peroxisomal ß-oxidation were induced dose-dependently by T0901317, we focused our attention on LXR regulation of the peroxisomal ß-oxidation pathway.

LXR regulation of the ß-oxidation pathway is independent of PPAR{alpha}
Because PPAR{alpha} has a profound effect on peroxisomally mediated ß-oxidation of lipids, and PPAR{alpha} also increases LXR gene expression, we explored whether LXR agonist induction of the ß-oxidation pathway is PPAR{alpha} dependent. Wild-type 129Sv mice exposed to 50 mg/kg T0901317 for 1 wk demonstrated a 2-fold increase in peroxisomal ß-oxidation, whereas treatment of wild-type mice with 300 mg/kg of the PPAR{alpha} agonist, fenofibrate, caused a greater than 7-fold response (Fig. 2AGo). In PPAR{alpha}-null mice, T0901317 also induced peroxisomal ß-oxidation by greater than 2-fold, and as anticipated, the PPAR{alpha} agonist, fenofibrate, showed no effect in the PPAR{alpha}-null mice (Fig. 2AGo). In a T0901317 dose-response experiment using PPAR{alpha}-null mice, both peroxisomal ß-oxidation and Acox and Ehhadh target genes were induced dose-dependently to levels similar to those observed in wild-type mice (Fig. 2Go, B and C). Although the Acaa1 gene was induced by T0901317, a dose-responsive induction profile was not observed. These data demonstrate that T0901317 induces hepatic peroxisomal ß-oxidation in mice independently of PPAR{alpha}.



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  		FIG. 2. LXR agonist induction of the ß-oxidation pathway occurs independently of PPAR{alpha} activity. A, Measurement of peroxisome-mediated ß-oxidation of lipids was performed using liver tissue from either 129Sv wild-type mice (WT) or PPAR{alpha}-null mice (PAKO) treated with 50 mg/kg T0901317 or with 300 mg/kg fenofibrate for 1 wk. Data are expressed as the mean ± SEM of five mice per treatment group. Statistical analysis using one-way ANOVA, followed by comparison with vehicle by Dunnett’s method, were performed. *, P ≤ 0.05. B, Measurement of peroxisome-mediated ß-oxidation of lipids was performed using liver tissue from PPAR{alpha}-null mice treated with T0901317 at the indicated doses for 1 wk. Data are presented as the mean ± SEM. Statistical analysis using one-way ANOVA, followed by comparison with vehicle by Dunnett’s method, were performed. *, P ≤ 0.05. C, Quantitative measurement of selected target gene transcripts by Q-RT PCR performed using RNA isolated from liver tissue of PPAR{alpha}-null mice treated with T0901317 at the indicated doses for 1 wk. Data were normalized to the 36B4 gene and are presented as the mean ± SEM. Statistical analysis using one-way ANOVA, followed by comparison with vehicle by Dunnett’s test, were performed. *, P ≤ 0.05.

 
LXR{alpha}, but not LXRß, regulates the liver ß-oxidation pathway
The two LXR isoforms, LXR{alpha} and LXRß, have diverse tissue distribution, but similar protein structures, and target DNA-binding elements and ligands. To investigate the involvement of each LXR isoform in peroxisomal ß-oxidation, isoform-specific ASO were used to knock down the expression of LXR in vitro and in vivo. Optimal ASO sequences were selected using a mouse primary hepatocyte model system transfected with a dose response of LXR{alpha} or LXRß ASOs. The extent of LXR{alpha} and LXRß target knockdown was assessed by Q-RT PCR. At 200 and 100 nM, respectively, LXR{alpha} and LXRß ASOs diminished the levels of LXR{alpha} and LXRß mRNAs in primary mouse hepatocytes by more than 80%, whereas the control ASO had no effect on either LXR{alpha} or LXRß mRNA levels at any concentration tested (Fig. 3AGo).



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  		FIG. 3. LXR ASOs decrease LXR{alpha} and LXRß mRNA in vitro and in vivo. A, Mouse primary hepatocytes were treated with the indicated ASO concentration for 4 h as described in Materials and Methods. After oligonucleotide treatment, cells were cultured for an additional 16–18 h. Total RNA was extracted, and LXR{alpha} and LXRß mRNA expression was assessed by RT-PCR. LXR expression was normalized to total RNA in the same samples using Ribogreen. Data are expressed as a percentage of the saline-treated control values. B, Control (Cont), LXR{alpha}, and LXRß ASOs were administered to C57BL/6 mice for 4 wk. All ASOs were administered via sc injection at 37.5 mg/kg. During the last week of ASO administration, either vehicle ({square}) or doses of 10 mg/kg T0901317 ({blacksquare}) were administered by oral gavage to the indicated groups of mice. Total liver RNA was extracted, and LXR{alpha} and LXRß mRNA expression was assessed by RT-PCR and corrected using the 36B4 gene. Data are expressed as the mean ± SEM of five mice per treatment group. Statistical analysis using one-way ANOVA, followed by comparison with control ASO mice treated with T0901317, followed by Dunnett’s method, were performed. *, P ≤ 0.05. C, Assessment of LXR target genes from livers of ASO-treated mice. Total liver RNA was extracted, and SREBP1, FASN, and Cyp7A1 mRNA expressions were assessed by RT-PCR and corrected using the 36B4 gene. Data are expressed as the mean ± SEM of five mice per treatment group. Statistical analysis using one-way ANOVA, followed by comparison with control ASO mice treated with T0901317, followed by Dunnett’s test, were performed. *, P ≤ 0.05.

 
Cohorts of mice were treated with these optimized isoform-selective ASOs every 3.5 d for 3 wk. While continuing the ASO treatment regimen, all groups were administered T0901317 (10 mg/kg, orally) for 1 wk. When administered to C57BL/6 mice, both LXR{alpha} ASO and LXRß ASO significantly reduced the expression of their respective target mRNA compared with levels from livers of control ASO-treated mice (Fig. 3BGo). These data confirm that the LXR{alpha} and LXRß ASOs are isoform specific and are efficacious in reducing target mRNAs in vivo. It is worth noting that the LXR{alpha} ASO is more potent in reducing target levels in vivo than the LXRß ASO (Fig. 3BGo). To confirm predicted functional outcomes of LXR{alpha} and LXRß knockdown by ASO technology, we also measured the expression of the well-characterized LXR target genes, SREBP1, FASN, and cholesterol 7{alpha}-hydroxylase (Cyp7a1). In mice treated with control ASO, T0901317 induced SREBP1 mRNA levels by more than 5-fold, FASN mRNA levels by more than 2-fold, and Cyp7a1 mRNA levels by 6-fold (Fig. 3CGo). Although both ASOs significantly reduced the induction of these target genes, the LXR{alpha} ASO was more efficacious in reducing levels of all target genes (Fig. 3CGo). This observation may be attributed to the relative higher potency of the LXR{alpha} ASO in reducing target levels compared with the LXRß ASO or to the prominence of LXR{alpha} as a key regulator of the hepatic lipogenic and bile acid synthetic gene programs.

The regulatory roles of LXR{alpha} and LXRß on the expression of genes involved in peroxisomal ß-oxidation were evaluated by both GeneChip analysis and Q-RT PCR using the same liver mRNA samples. As determined by microarray analysis and summarized in Table 1Go, ASO ablation of LXR{alpha} expression resulted in reduced expression of the three peroxisomal ß-oxidation genes, Acox, Ehhadh, and Acaa1. Ablation of LXRß expression reduced Acaa1 only. Confirmatory Q-RT PCR analysis demonstrated that T0901317-induced expressions of Acox, Ehhadh, and Acaa1 were completely abolished by LXR{alpha} ASO treatment. In contrast, the expressions of both Ehhadh and Acaa1 genes in LXRß ASO-treated mice were identical with control ASO levels (Fig. 4AGo). Only T0901317 induction of the Acox gene was significantly reduced by LXRß ASO administration. That the LXR{alpha} isoform primarily regulates ß-oxidation was confirmed in the liver ß-oxidation assay. T0901317 stimulated peroxisomal ß-oxidation in both control and LXRß ASO-treated mice, but not in LXR{alpha} ASO-treated mice (Fig. 4BGo). In total, these results indicate that LXR-induced peroxisomal ß-oxidation is dependent on the LXR{alpha} isoform, but not the LXRß isoform.



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  		FIG. 4. LXR{alpha} ASO selectively reduces LXR agonist-mediated induction of liver ß-oxidation of lipids. A, Control (Cont), LXR{alpha}, and LXRß ASOs were administered to C57BL/6 mice for 4 wk. All ASOs were administered via sc injection at 37.5 mg/kg. During the last week of ASO administration, either vehicle ({square}) or doses of 10 mg/kg T0901317 ({blacksquare}) were administered by oral gavage to the indicated groups of mice. Total liver RNA was extracted, and mRNA expression was assessed by RT-PCR and corrected using the 36B4 gene. Data are expressed as the mean ± SEM of five mice per treatment group. Statistical analysis using one-way ANOVA, followed by comparison with control ASO mice treated with T0901317, followed by Dunnett’s method, were performed. *, P ≤ 0.05. C, Rates of ß-oxidation were measured, as described in Materials and Methods, from liver tissue of the mice treated as described in A. Data are expressed as the mean ± SEM of five mice per treatment group. Statistical analysis using one-way ANOVA, followed by comparison with control ASO mice treated with T0901317, followed by Dunnett’s test, were performed. *, P ≤ 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
As a transcriptional regulator of genes such as the ABC transport proteins, apolipoprotein E, lipoprotein lipase, SREBP, FASN, and cholesterol 7{alpha}-hydroxylase, LXR has emerged as key transcription factor regulating reverse cholesterol transport from the periphery, increasing hepatic cholesterol catabolism to bile acids, and lipogenesis (5, 6, 7, 9, 11, 12, 13, 14, 16). While investigating the pharmacology of LXR agonists in liver, we found that not only does LXR increase lipid synthesis by inducing the expression of genes such as fatty acid synthase, but it also increases the catabolism of fatty acids via induction of peroxisomal ß-oxidation genes in liver. The expressions of all three enzymes of the classic peroxisomal ß-oxidation cycle, acyl-CoA oxidase, enoyl-CoA hydratase/L-3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase, were activated by the LXR ligand, T0901317. Notably, known target genes of LXR in the liver, such as SREBP1c, FASN, phospholipid transfer protein (5, 6, 7, 8, 22), and apolipoprotein AIV (23), were induced in the microarray experiment as expected (data not shown). Although studies of genome-wide expression analyses from T0901317-treated mice report regulation of genes involved in fatty acid catabolism by LXR in liver and adipose (24, 25), our study is the first to confirm the target gene regulation, to characterize the isoform specificity of the regulation, and to measure changes in rates of hepatic peroxisomal ß-oxidation in response to an LXR agonist.

For more than 30 yr, peroxisome proliferator molecules have been linked to increased hepatic lipid ß-oxidation activity and decreased plasma triglyceride levels in multiple rodent species. The molecular mechanism of action of these molecules on lipid oxidation occurs principally via activation of PPAR{alpha}. We now describe an alternative mechanism by which the peroxisomal lipid ß-oxidation pathway may be induced through activation of LXR{alpha}. We find that the LXR agonist, T0901317, stimulates peroxisomal ß-oxidation in the liver. The induction of ß-oxidation activity as well as the expression of the enzymes in the pathway are induced 2-fold, which is modest by comparison to the 7-fold induction promoted by PPAR{alpha} agonists. The LXR agonist induced peroxisomal ß-oxidation to nearly the same extent in livers of wild-type and PPAR{alpha}-null mice, demonstrating that PPAR{alpha} is not required for LXR-mediated increases in lipid ß-oxidation.

Regulation of the genes involved in peroxisomal ß-oxidation by PPAR agonists occurs through a direct transcriptional response. After activation by either synthetic ligands or specific fatty acid derivatives, PPAR{alpha} binds to a DNA response element, termed the peroxisome proliferator-responsive element, located in the promoter region of the target genes. The representative cognate peroxisome proliferator-responsive element consists of a direct repeat of the consensus sequence, TGACCT, spaced by one nucleotide and is present in the majority of genes involved in peroxisomal ß-oxidation (26). LXRs bind to cognate LXR response element (LXRE) sequences that typically consist of a direct repeat of TGACCT spaced by four nucleotides, or DR-4 (27, 28). Comparative analysis and inspection of 5 kb upstream of the transcription start site of all three genes from both mouse and human genomes revealed at least one putative LXRE in each gene (data not shown). Whether these DR-4 elements serve as bona fide LXREs that mediate direct transcriptional regulation by LXR{alpha} remains to be determined. The mechanism underlying the changes in peroxisomal ß-oxidation gene expression in response to LXR activation also may be indirect, secondary to the direct effects of LXR on lipogenesis. Because T0901317 induces Acox, Ehhadh, or Acaa1 gene transcription in mice lacking PPAR{alpha}, it is unlikely that the LXR agonist induces ß-oxidation through the generation of endogenous lipid ligands that activate PPAR{alpha}. Whether other transcription factors, such as SREBP-1, that may act subsequent to LXR activation are involved in modulating peroxisomal ß-oxidation gene expression is, as yet, undetermined.

Through the use of LXR isoform-selective ASOs, we demonstrate that the induction of peroxisomal ß-oxidation by T0901317 is dependent on LXR{alpha}, but not LXRß. In contrast, the induction of known LXR target genes, such as SREBP1, FASN, and Cyp7a1, was significantly reduced by both the LXR{alpha}- and LXRß-specific ASOs. The rate of hepatic ß-oxidation and the levels of Acox and Ehhadh expression were induced 2-fold by T0901317 in normal mice and LXRß ASO-treated mice. However, the induction was largely lost in mice lacking LXR{alpha} expression. That only LXR{alpha} is required for T0901317-mediated induction of the peroxisomal ß-oxidation program supports the concept that despite their conserved protein structure, the LXRs maintain distinct biological roles (16, 29). Similar results were observed in LXR-mediated regulation of lipoprotein lipase hepatic gene expression; LXR{alpha} selectively increases LPL expression (16). Whether synthetic LXR{alpha} ligands can be developed to not only increase the reverse cholesterol transport system, but also to potentiate genes involved in ß-oxidation to diminish LXR-induced hypertriglyceridemia, is currently unknown.


   Footnotes
 
First Published Online August 25, 2005

Abbreviations: Acaa1, 3-Ketoacyl-coenzyme A thiolase; Acox, acyl-coenzyme A oxidase; ASO, antisense oligonucleotide; CoA, coenzyme A; Ehhadh, enoyl-coenzyme A hydratase/L-3-hydroxyacyl-coenzyme A dehydrogenase; FASN, fatty acid synthase; LXR, liver X receptor; LXRE, LXR response element; PPAR{alpha}, peroxisome proliferator-activated receptor {alpha}; Q-RT, quantitative real-time; SREBP1, sterol response element-binding protein 1c; VLCF, very-long-chain fatty acid.

Received May 16, 2005.

Accepted for publication August 15, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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